Compositions and methods for detection and isolation of phosphorylated molecules

ABSTRACT

The present invention relates to phosphate-binding compounds that find use in binding, detecting and isolating phosphorylated target molecules including the subsequent identification of target molecules that interact with phosphorylated target molecules or molecules capable of being phosphorylated. A binding solution is provide that comprises a phosphate-binding compound, an acid and a metal ion wherein the metal ion simultaneously interacts with an exposed phosphate group on a target molecule and the metal chelating moiety of the phosphate-binding compound forming a bridge between the phosphate-binding compound and a phosphorylated target molecule resulting in a ternary complex. The binding solution of the present invention finds use in binding and detecting immobilized and solubilized phosphorylated target molecules, isolation of phosphorylated target molecules from a complex mixture and aiding in proteomic analysis wherein kinase and phosphatase substrates and enzymes can be identified.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/703,816, filedNov. 7, 2003 which is a continuation-in-part of U.S. Ser. No.10/428,192, filed May 2, 2003 which claims priority to U.S. Ser. No.60/377,733, filed May 3, 2002; U.S. Ser. No. 60/393,059, filed Jun. 28,2002; U.S. Ser. No. 60/407,255, filed Aug. 30, 2002; and U.S. Ser. No.60/440,252, filed Jan. 14, 2003, which disclosures are hereinincorporated by reference. Any disclaimer that may have occurred duringthe prosecution of the above-referenced application(s) is herebyexpressly rescinded.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made in part with government support under grantnumber 1 R33 CA093292-01, awarded by the National Cancer Institute. TheUnited States Government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to metal-chelating compositions andmethods for use in the detection and isolation of phosphorylated targetmolecules. The invention has applications in the fields of proteomics,molecular biology, high-throughput screening and diagnostics.

BACKGROUND OF THE INVENTION

Phosphorylation and dephosphorylation are processes by which phosphategroups are added or removed from a target molecule, typically a protein.The process of reversible phosphorylation is a key feature of cellularregulation, including signal transduction, gene expression, cell cycleregulation, cytoskeletal regulation and apoptosis. See, e.g., PROTEINPHOSPHORYLATION (Marks F. ed., 1996); Hunter, “Signaling—2000 andbeyond,” Cell 100:113-127 (2000). Principally, two classes of enzymes(kinases and phosphatases) modulate reversible protein phosphorylation,adding phosphate groups and removing phosphate groups, respectively,from molecules. Phosphorylation reactions are key features of proteinfunction, and thus phosphorylated proteins must be able to be identifiedif the proteome is to be fully understood; however, to date no practicalmethods exist for the systematic parallel analysis of thephosphorylation status of large sets of proteins involved in theregulatory circuitry of cells and tissues. See, Wilkins et al., GeneticEng. Rev. 13:19 (1995).

Signal transduction is an example of a process involving proteinphosphorylation that is critical for cellular regulation. After anextracellular stimulatory factor binds to its recognized cell surfacereceptor, signal transduction is initiated, often by a specific set ofcellular protein kinases. These kinases subsequently phosphorylate thetarget molecule, resulting in an altered activity and a continuedcellular response to the signal. See, e.g., Nishizuka, “Studies andperspectives of protein kinase C,” Science 233:305-312 (1986). It is notenough for researchers to simply identify whether a protein is aphosphorylated protein or not. It has become additionally essential forresearchers to identify the sites of phosphorylation on proteins and todetermine the stoichiometry of phosphorylation. Serine, threonine andtyrosine amino acid residues are the most common sites ofphosphorylation in eukaryotic cells. See, e.g., Guy et al. “Analysis ofCellular Phosphoproteins by Two-Dimensional Gel Electrophoresis:Applications for Cell Signaling in Normal and Cancer Cells,”Electrophoresis 15:417-440 (1994). Thus, the focus for researchers inunderstanding protein phosphorylation events occurs at two levels. Thefirst level of analysis requires a determination of whether a protein isa phosphoprotein, including identifying molecules responsible forphosphorylation, and the second level of analysis requires theidentification of which amino acid is phosphorylated and how many aminoacids are phosphorylated. The present invention provides materials andmethods for both levels of analysis. The present invention also providesmaterials and methods for analysis of certain other phosphate andthiophosphate-containing materials including esters of carbohydrates,nucleotides and lipids.

Currently, phosphoproteins are most often detected by autoradiographyafter incorporation of ³²P or ³³P into cultured cells or afterincorporation into subcellular fractions by protein kinases. See, e.g.,Yan et al., “Protein Phosphorylation: Technologies for theIdentification of Phosphoamino Acids,” J. Chromatogr. A. 808:23-41(1998); Guy, G., Phillip, R. and Tan, Y. Electrophoresis 15:417-440(1994). Such approaches are restricted to a limited range of biologicalmaterials, such as tissue culture samples and analysis of clinicalsamples would require in vivo labeling of patients, which is notfeasible. Several alternatives to radiolabeling have also been developedover the years.

Phosphoproteins can also be detected by immunoblotting andimmunoprecipitation. See, e.g., Soskic et al., “Functional ProteomicsAnalysis of Signal Transduction Pathways of the Platelet-Derived GrowthFactor Beta Receptor,” Biochemistry 38:1757-1764 (1999); Watty et al.,“The In Vitro and In Vivo Phosphotyrosine Map of Activated MuSK,” Proc.Natl. Acad. Sci. USA. 97:4585-4590 (2000). Immunoblotting is bestperformed after blocking unoccupied sites on the solid-phase supportwith protein solutions, which interferes with microchemical analysis.Removal of the antibody and stain require relatively harsh treatments(i.e., heating to 65° C., incubation with 0.1% SDS and 1 mM DTT). Thisalso poses problems with subsequent use of the protein for sequencingand mass spectrometry. For immunoprecipitation, only theanti-phosphotyrosine antibodies display binding that is tight enough toallow effective isolation. Though high-quality antibodies tophosphotyrosine are commercially available, antibodies that specificallyrecognize phosphoserine and phosphothreonine residues have been moreproblematic, often being sensitive to amino acid sequence context. Thereliability of these antibodies has been questioned because of potentialsteric hindrances between the interaction of these antibodies and thephosphoproteins. Moreover, when phosphoproteins are not enriched priorto detection with the antibody, the presence of unrelated proteinsco-migrating with the protein of interest may lead to false positivesignals. Therefore, identification of phosphorylated proteins usingimmunoblotting and immunoprecipitation techniques is effectively limitedto proteins containing phosphorylated tyrosine residues. See McLachlin &Chait, supra.

Alternatively, phosphorylated proteins can be identified usingchromogenic dyes, but with limited success. The cationic carbocyaninedye “Stains-All”(1-ethyl-2-[3-(3-ethylnaphtho[1,2d]thiazolin-2-ylidene)-2-methylpropenyl]-naphtho[1,2d]thiazoliumbromide) stains RNA, DNA, phosphoproteins and calcium-binding proteinsblue while unphosphorylated proteins are stained red. See, e.g., Greenet al., “Differential Staining of Phosphoproteins on Polyacrylamide Gelswith a Cationic Carbocyanine Dye,” Anal. Biochem. 56:43-51 (1973);Hegenauer et al., “Staining Acidic Phosphoproteins (Phosvitin) inElectrophoretic Gels,” Anal. Biochem. 78:308-311 (1977); Debruyne,“Staining of Alkali-Labile Phosphoproteins and Alkaline Phosphatases onPolyacrylamide Gels,” Anal. Biochem. 133:110-115 (1983); “Staining ofphosphoproteins in polyacrylamide gels with acridine orange”, Seikagaku45:327-35 (1973). Stains-All is not routinely used to detectphosphoproteins due to poor specificity and low sensitivity. Stains-Allis at least 10 times less sensitive than Coomassie Brilliant Blue as ageneral protein stain and several orders of magnitude less sensitivethan ³²P-autoradiography or the techniques described in this patent.Another chromogenic method, the GelCode™ Phosphoprotein detection kit(Pierce Chemical Company, Rockford, Ill.), is designed to detectphosphoproteins in gels; however, this method has many limitations.According to this method, phosphoproteins are detected in gels throughalkaline hydrolysis of phosphate esters of serine or threonine,precipitation of the released inorganic phosphate with calcium ions,formation of an insoluble phosphomolybdate complex and thenvisualization of the complex with a dye such as methyl green, malachitegreen or rhodamine B [as described in Cutting and Roth (1973)]. Thedetection sensitivity of the staining method is considerably poorer thanCoomassie Blue staining, with 80-160 ng of phosvitin, a proteincontaining roughly 100 phosphoserine residues, being detectable by thecommercialized kit. The staining procedure is quite complex (involvingseven different reagents) and alkaline hydrolysis requires heating gelsto 65° C., which causes the gel matrix to hydrolyze and swellconsiderably. Since phosphotyrosine residues are not hydrolyzed by thealkaline treatment, proteins phosphorylated at this amino acid residueescape detection by the method. Dyes for the phosphate-selectivefluorescence labeling in which a BODIPY dye is covalently attached to areactive imidazole group has been developed for the detection of pepsinphosphorylation. See, U.S. Pat. No. 5,512,486; Wang & Giese,“Phosphate-Specific Fluorescence Labeling of Pepsin by BO-IMI,” Anal.Biochem. 230:329-332 (1995).

In addition to detecting phosphoproteins, two methods for the chemicalderivatization and enrichment of phosphopeptides resulting in isolationof phosphopeptides from complex mixtures exist. See, e.g. Goshe et al.,“Phosphoprotein Isotope-Coded Affinity Tag Approach For Isolating andQuantitating Phosphopeptides in Proteome-Wide Analyses,” Anal. Chem.73:2578-2586 (2001). The first method involves oxidation of cysteineresidues with performic acid, alkaline hydrolysis to induceβ-elimination of phosphate groups from phosphoserine andphosphothreonine residues, addition of ethanedithiol, coupling of theresulting free sulfhydryl residues with biotin, purification ofphosphoproteins by avidin affinity chromatography, proteolytic digestionof the eluted phosphoproteins, a second round of avidin purification andthen analysis by mass spectrometry (Oda, Y., Nagasu, T., and Chait, B.Nature Biotechnol. 19:379 (2001)). The first method uses β-eliminationto remove phosphate groups that are replaced with a tag, as exemplifiedwith biotinylated thiol groups wherein the peptides could then beisolated by chromatography on avidin resins. An alternative methodrequires proteolytic digestion of the sample, reduction and alkylationof cysteine residues, N-terminal and C-terminal protection of thepeptides, formation of phosphoramidate adducts at phosphorylatedresidues by carbodiimide condensation with cystamine, capture of thephosphopeptides on glass beads coupled to iodoacetate, elution withtrifluoroacetic acid and evaluation by mass spectrometry (Zhou et al.,“A Systematic Approach to the Analysis of Protein Phosphorylation,” Nat.Biotechnol. 19:375-378 (2001). These methods are time consuming, requirepurified phosphopeptides, and are limiting in what can be isolated. Bothprocedures identified the monophosphorylated trypsin peptide fragmentfrom the test protein β-casein, but both failed to detect thetetraphosphorylated peptide fragment.

Alternatively, a method for combining chemical modification and affinitypurification has been shown for the characterization of serine andthreonine phosphopeptides in proteins based on the conversion ofphosphoserine and phosphothreonine residues toS-(2-mercaptoethyl)cysteinyl or β-methyl-S-(2-mercaptoethyl)cysteinylresidues by β-elimination/1,2-ethanedithiol addition, followed byreversible biotinylation of the modified proteins. After trypsindigestion, the biotinylated peptides are affinity-isolated and enriched,followed by their subsequent structural characterization by liquidchromatography/tandem mass spectrometry (LC/MS/MS). See Adamczyk et al.,“Selective Analysis of Phosphopeptides Within a Protein Mixture byChemical Modification, Reversible Biotinylation and Mass Spectrometry,”Rapid. Commun. Mass Spectrom. 15:1481-1488 (2001).

Fluorescence detection methods appear to offer the best solution toglobal protein quantitation in proteomics. However, currently, there isno satisfactory method for the specific and reversible fluorescentdetection of gel-separated phosphoproteins from complex samples.Derivatization and fluorophore labeling of phosphoserine residues byblocking free sulfhydryl groups with iodoacetate or performate, alkalineβ-elimination of the phosphate residue, addition of ethanedithiol, andreaction of the resulting free sulfhydryl group with6-iodoacetamidofluorescein has been demonstrated in capillaryelectrophoresis using laser-induced fluorescence detection and similarreactions have been performed on protein microsequencing membranes.However, neither method has been shown to be suitable for detection ofphosphoproteins directly in gels. One problem with the approach is thata delicate balance must be struck between the base and the ethanedithiolin order to achieve elimination of the phosphate group from the serineresidue and addition of the ethanedithiol to the resultingdehydroalanine residue without hydrolysis of the peptide backbone.

Several instrument-based methods are also available for thedetermination of protein phosphorylation such as ³¹P-NMR, massspectrometry [See, e.g., Resing & Ahn, “Protein Phosphorylation Analysisby Electrospray Ionization-Mass Spectrometery,” Methods Enzymol.283:29-44 (1997); Aebersold and Goodlett, “Mass Spectrometry inProteomics,” Chem. Rev. 101:269-295 (2001). Affolter, M., Watts, J.,Krebs, D., and Aebersold, R. Anal. Biochem. 223:74 (1994); Liao, P.,Leykam, J., Andrews, P., Gage, D., and Allison, J. Anal. Biochem. 219:9(1994); Oda, Y., Huang, K., Cross, F., Cowburn, D., and Chait, B. Proc.Natl. Acad. Sci. USA 96:6591 (1999)) and protein sequencing. Massspectrometry has been used to provide the molecular mass of an intactphosphorylated protein by comparing the mass of the unphosphorylatedprotein to that of the phosphorylated protein. See, e.g., McLachlin &Chait, “Analysis of Phosphorylated Proteins and Peptides by MassSpectrometry,” Current Opin. Chem. Biol. 5:591-602 (2001). This islimiting in that researchers must have purified amounts of bothproteins. While these procedures accurately characterize thephosphorylation status of proteins and peptides, they are unsuitable forhigh-throughput screening of phosphorylated substrates. The techniquesare generally used after a phosphoprotein has been identified byautoradiography or immunoblotting with an anti-phosphotyrosine antibody.Though methods have recently been introduced to directly quantify therelative abundance of phosphoproteins in two different samples by massspectrometry through culturing different cell populations in¹⁵N-enriched and ¹⁴N-enriched medium, the linear dynamic range of suchmethods has explicitly been demonstrated over only a 10-fold range. Ionsuppression phenomena associated with mass spectrometry preventsstoichiometric comparison of different phosphoproteins by suchtechniques.

For analysis of the site(s) of phosphorylation on molecules, a moredetailed analysis of the sites of phosphate attachment and stoichiometryoften requires the examination of peptide fragments of thephosphoprotein of interest. Such fragments are usually generated bydigestion of the phosphoprotein with proteases such as trypsin. However,mass spectroscopic analysis of proteolytic digests of proteins rarelyprovides full coverage of the protein sequence and regions of interestsoften go undetected. In addition, protein phosphorylation is oftensub-stoichiometric, such that the phosphoproteins are present in lowerabundance than other peptides from the protein of interest. Therefore,the identification and characterization of phosphoproteins would beimproved greatly by highly selective methods of enrichingphosphopeptides prior to analysis with mass spectrometry. It would beparticularly useful to detect phosphoproteins by reagents that do notchemically alter the structure or mass of the phosphoproteins.

Currently, selective enrichment of phosphopeptides from complex mixturesis performed using immobilized metal affinity chromatography, known asIMAC. Using this technique, metal ions such as Fe³⁺ or Ga⁺³ are bound toa chelating support prior to the addition of a complex mixture ofpeptides or proteins. See, e.g., Posewitz & Tempst, “ImmobilizedGallium(III) Affinity Chromatography of Phosphopeptides,” Anal. Biochem.71:2883-2892 (1999). Phosphopeptides that bind to the column can bereleased using high pH or phosphate buffer, though the latter stepusually requires a further desalting step before analysis with massspectrometry. Resins with iminodiacetic acid and nitrilotriacetic acidchelators are known and are available commercially. See, e.g., Nevilleet al., “Evidence for Phosphorylation of Serine 753 in CFTR Using aNovel Metal-Ion Affinity Resin and Matrix-Assisted Laser Desorption MassSpectrometry,” Protein Sci. 6:2436-2445 (1997). However, there areseveral complications using current techniques, including loss ofphosphopeptides that do not bind to the column (low affinity),difficulty in the subsequent elution of phosphorylated peptides, andbackground from non-phosphorylated peptides that have affinity forimmobilized metal ions (low specificity).

Mass spectrometry-based detection of separated peptides and directmatrix-assisted laser desorption/ionization (MALDI) analysis ofphosphopeptides bound to an IMAC support has been demonstrated. See Zhouet al., “Detection and Sequencing of Phosphopeptides Affinity Bound toImmobilized Metal Ion Beads by Matrix-Assisted LaserDesorption/lonization Mass Spectrometry,” J. Am. Soc. Mass. Spectrom.11:273-283 (2000). IMAC has also been coupled directly to massspectrometry instruments on-line, or with superseding separationtechniques, such as HPLC and capillary electrophoresis (CE), for thedetection and analysis of phosphopeptides.

The present invention overcomes the limitations of the current methodsby utilizing a cationic transition metal and a compound that comprises ametal-chelating moiety and a chemical moiety, typically a reactive groupor label such as a fluorophore, or a combination thereof, to detectphosphoproteins and phosphopeptides. There are a variety of chelatingmoieties that use poly-carboxylate binding sites to selectively bindmonovalent and divalent metal cations, and these are often used influorescent calcium ion indicators. Examples of these indicators are,for example, quin-2, fura-2, indo-1 (U.S. Pat. No. 4,603,209); fluo-3and rhod-2 (U.S. Pat. No. 5,049,673), and FURA RED™ (U.S. Pat. No.4,849,362). A family of BAPTA-based indicators that are selective forcalcium ions are described in HAUGLAND, HANDBOOK OF FLUORESCENT PROBESAND RESEARCH CHEMICALS (9 ^(th) edition, CD-ROM, September 2002).Examples of BAPTA-based metal-chelators are also described in U.S. Pat.Nos. 5,773,227; 5,453,517; 5,516,911; 5,501,980; 6,162,931 and5,459,276.

Indicators of free calcium concentrations are based upon selectivecalcium binding to fluorescent dyes. Though it is well known that BAPTAcompounds bind certain divalent cations, such as calcium, as analogs ofthe common EGTA chelator, the BAPTA compounds are also known to bindalmost all inorganic polyvalent cations with an affinity that may behigher or lower than that of the compound for calcium ions. Theirselectivity and utility for measuring calcium in biological cellsresults from the general absence or low abundance of these otherpolycations in living systems. The affinity and selectivity of theBAPTA-based indicators for polycations, including gallium and similarmetals of utility to this invention, can be modified by shifts in pH,solvent composition, ionic strength and other experimental variables.This shift in cation selectivity and affinity is critical to all aspectsof the disclosed invention, including both detection and isolation ofphosphorylated targets.

The present invention overcomes the limitations and disadvantages ofcurrently disclosed methods for identifying, isolating, analyzing andquantitating phosphorylated proteins and thus provide methods, compoundsand compositions to alleviate long-felt needs for rapid and effectivehigh-throughput methods for detecting and isolating phosphoproteins forfurther analysis. The present invention can accurately identifyphosphopeptides and phosphoproteins comprising as few as one phosphategroup and in a simple method that does not require multiple steps orpre-treatment of the sample. Importantly, the present invention is thefirst known method to provide a means for accurately identifying thephosphorylated proteome and allows for the quantitative identificationof increased phosphorylation of proteins. The present invention is animportant tool for identifying novel phosphorylated proteins in theproteome. The technology has unsurpassed quantitative characteristics,particularly when used in combination with reagents for the detection oftotal proteins. In addition, as will be described below, the materialsand methods of the present invention are not limited to the detectionand/or separation of phosphorylated proteins.

SUMMARY OF THE INVENTION

The present invention provides phosphate-binding compounds and methodsfor specifically detecting, isolating and/or quantitating phosphorylatedtarget molecules. These compounds, when in a moderately acidicenvironment, and in the presence of an appropriate metal ion willselectively bind phosphate groups on phosphorylated target moleculesthat are either immobilized, such as in a gel, or adsorbed on a solid orsemisolid matrix, or are dissolved or suspended in a mostly aqueoussolution to form a ternary complex.

The phosphate-binding compounds comprise a metal chelating moiety thatis optionally bonded to a label or a chemically reactive group. Thus, inone aspect the phosphate-binding compounds have the formula (A)m(L)n(B),wherein A is a chemical moiety, L is a linker, B is a metal-chelatingmoiety, m is an integer from 1 to 4 and n is an integer from 0 to 4.Without being bound by theory, it appears that the metal-chelatingmoiety of these particular phosphate-binding compounds simultaneouslybinds a trivalent metal ion and a phosphate group on a target moleculein a reaction that forms a ternary complex. In this way, the metal ionprovides a bridge between the phosphate group and the metal-chelatingmoiety wherein the chemical moiety A is effectively bound to thephosphate target molecule by ionic interactions. Thus, it is arequirement of the present invention that the metal-chelating moietybind a metal ion that has simultaneous affinity for the phosphorylatedtarget molecule, under appropriate conditions.

The utility of the compositions and methods of this invention isprincipally the result of the chemical moieties A that are covalentlyattached to the metal-chelating moieties by a linker to form thephosphate binding compounds of the present invention. Typically,chemical moieties A are a reactive group or a label. The reactive groupsfunction to covalently attach another natural or synthetic substance tothe metal-chelating moieties or alternatively covalently bind thephosphorylated target molecule after the metal-chelating moiety andmetal ion has brought the reactive group in close proximity to thephosphorylated target molecule. Particularly useful substances that thereactive group A covalently binds to the metal chelating moiety offormula (A)m(L)n(B) include without limitation particles, polymers,peptides and proteins. In this way, a particle could have manyphosphate-binding compounds attached.

For detection purposes, A is typically a detectable label that is a dyeincluding pigments, chromophores and fluorophores, haptens, enzymes, orradioactive isotopes, although an extensive assortment of otherdetectable labels that fall within the scope of this invention is known.For isolation purposes of phosphate containing targets, A is typically alabel or a reactive group that is bound to a polymer such as agarose, asurface, a magnetic particle or a microsphere. The polymer, incombination with the metal chelating moiety and a metal ion is selectedto form a soluble or insoluble ternary complex with the phosphorylatedtarget molecule. Such ternary complexes are particularly useful for theselective isolation of phosphorylated targets from complex mixtures oras components of various detection schemes. In a further aspect of theinvention, A is a chemical moiety that alters the solubility of theternary complex or alternatively comprises an amine-reactive group usedto form a covalent bond with an amine-containing molecule, includingpolymers and phosphate target molecules.

The “binding solution” of the invention (which we define to include truesolutions, suspensions, emulsions, dispersions and immobilized variantsthereof) of the present invention comprises a phosphate-bindingcompound, typically having the formula (A)m(L)n(B), a salt comprisingselected metal ions, and an acid. The preferred salt and metal ioncomposition and concentration of the binding solution or suspension willdepend to some extent on the metal-chelating moiety of the compound. Aparticularly useful binding solution is the combination of a BAPTA-basedchelating moiety of the phosphate-binding compound and a gallium salt.Unexpectedly, we have determined that trivalent gallium ionssimultaneously bind BAPTA moieties and phosphorylated target moleculesto form a ternary complex with a useful affinity only in the presence ofa moderately acidic environment. However we have also shown, othermetal-chelating moieties such as DTPA, IDA and phenanthroline tosimultaneously bind gallium trivalent ions and phosphate groups. Thus,one requirement of the binding solution is the presence of an acid,wherein the binding solution preferably has a pH of about 3 to about 6;typically the pH is about 3 to about 4. The nature of the acid used toobtain this pH appears to be irrelevant; however, a phosphoric acid,phosphonic acid or polyphosphoric acid should not be used to obtain thispH, as they could reduce the stability of the ternary complex.Typically, the phosphate-binding compound is free in the bindingsolution or suspension; however, the phosphate-binding compound can beimmobilized on a solid or semi-solid matrix such that when the metal ionand acid are added a binding solution is formed and a ternary complex ofthe invention is subsequently formed if a phosphorylated target ispresent in a sample.

The methods of the invention comprise contacting a sample with a bindingsolution comprising the phosphate-binding compound, the metal ion andthe acid, incubating the sample and the binding solution for sufficienttime to allow the compound of the binding solution to associate withsaid phosphorylated target molecule whereby said phosphorylated targetmolecule forms a ternary complex.

Typically, for detection purposes, the resulting ternary complex thatcomprises the compound is illuminated to measure a detectable opticalproperty of the chemical moiety A, whereby the presence of thephosphorylated target molecules is detected. The phosphorylated targetmolecules can be detected in solution or when immobilized on a solid orsemi-solid matrix. The compositions and methods of this invention can beused to detect phosphorylated target molecules present in a complexsample of phosphorylated and nonphosphorylated target molecules or todetect a change in the number of phosphate groups on a target molecule.Differences in the degree of phosphorylation can be due to intrinsicdifferences in the degree of phosphorylation of the biopolymer, whichcan cause differences in folding of proteins, or to an in vivo processsuch as a disease state or in conjunction with an in vitro assay toidentify specific kinases and phosphatases.

Alternatively, when the method is utilized to selectively isolatephosphorylated target molecules from solution, visualizing the complexmay not be necessary. To isolate the phosphorylated target molecules,the ternary complex can be precipitated, immobilized, separated by achromatographic or electrophoretic technique or by a magnetic field orremain in solution. In some cases, organic extraction can be used toseparate the metal-chelating moiety from the phosphorylated targetmolecule. When the ternary complex is precipitated or otherwiseimmobilized, the phosphorylated target molecules can be separated fromthe nonphosphorylated target molecules and other components of thesample by affinity chromatography, such as by simple washing with anaqueous, organic or mixed aqueous/organic wash solution. In some cases,it is advantageous to further analyze the extracted phosphorylatedtarget molecules while they are still immobilized on a matrix. Isolationof phosphorylated target molecules is useful for further analysis of thetarget molecules, such as by liquid chromatography/mass spectrography,an electrophoretic separation technique, by detection of the boundtarget molecules by an antibody to any part of the target molecule or bya variety of other techniques. In particular, isolation of thephosphorylated target molecules simplifies the subsequent analysis ofthe sample by removing interfering components of the original sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows the selective detection of a phosphorylated targetmolecule (ovalbumin) (1A) in a polyacrylamide gel using a bindingsolution comprising gallium chloride and Compound 2 compared to the samegel post-stained with a total protein stain (1B) (SYPRO® Ruby proteingel stain) (See, Example 2). The protein mixture was loaded at ca. 500μg and contained nine total proteins, one of which was a phosphoprotein(ovalbumin) that contains two phosphate groups. The figure demonstratesselective detection of ovalbumin against a background of very low or nostaining of eight proteins known to be non-phosphorylated.

FIG. 2: Shows the selective detection of a phosphorylated targetmolecule (ovalbumin) (2A) on a PVDF membrane using a binding solutioncomprising gallium chloride and Compound 1 compared to the same membranepost-stained with a total protein stain (2B) (SYPRO® Ruby protein blotstain) (See, Example 8). The figure demonstrates selective detection ofovalbumin against a background of very low or no staining of fivenon-phosphorylated proteins.

FIG. 3: Shows the sensitivity and linear dynamic range of detectingphosphorylated proteins in a gel using a binding solution comprisinggallium chloride and Compound 2 (See, Example 2); (3A) is a comparisonof five proteins with different ratios of phosphate groups and (3B)compares pepsin to bovine serum albumin (BSA). Proteins were loaded intwo-fold dilution series on SDS polyacrylamide mini-gels from 2 ng-1000ng; each protein sample was done in series in four replicate gels. Thephosphoproteins were α-casein (7 or 8 phosphates); dephosphorylatedα-casein (1 or 2 phosphates); β-casein (5 phosphates); ovalbumin (2phosphates) and pepsin (1 phosphate). BSA contains no phosphates and wasused as a negative control. The results demonstrate that the methods andbinding solution of the present invention can detect as little as 1-2 ngof a pentaphosphorylated protein (β-casein), and 8 ng of amonophosphorylated protein (pepsin).

FIG. 4: Shows the detection of protein phosphatase activity whereinα-casein and pepsin were used as a phosphatase substrate. The gels wereincubated with a binding solution comprising gallium chloride andCompound 2 to demonstrate a reduction in phosphate groups, compared to acontrol, after the substrates were incubated with a protein phosphatase,See example 6.

FIG. 5: Shows the isolation of phosphopeptides (pT/pY and RII; MWs 1670and 2193) (Panel B and C) from a solution containing non-phosphorylatedpeptides (angiotensin I and II, MWs 1297 and 1046) (Panel A) when thesolution of peptides was incubated with a binding solution comprisinggallium chloride and Compound 5. The mixture was incubated for 1 hourand centrifuged for 5 minutes. The resulting supernatants (bottomspectra in all panels) and pellet precipitates (top spectra in allpanels) were analyzed by MALDI-TOF mass spectrometry. Panel A shows thenon-phosphorylated peptides exclusively in the supernatants, whilefigures B and C show the two phosphopeptides of greater than 95% purityin the pellets.

FIG. 6: Shows the analysis of phosphorylated peptides (α-casein) elutedfrom an affinity chromatography matrix column containing Compound 13 orCompound 14 that had been charged with gallium ions. Panel A showsdifferential MALDI-TOF MS analysis of purified α-casein phosphoserinepeptides after dephosphorylation (left peaks in pairs) and subsequentderivatization with methylamine (right peaks in pairs). Results showthat all three peptides are phosphoserine derivatives by methylamineaddition. A and B of Panel A were monophosphorylated (+31 amu formethylamine) and C was triphosphorylated (+93 amu for 3 methylamines).Panel B shows a MALDI-TOF MS profile of eluted phosphopeptides from aBAPTA-agarose (Compound 13 or Compound 14) column versus commerciallyavailable metal affinity columns (Pierce Chemical Co.). Under theconditions used, the BAPTA-agarose column shows all expectedphosphopeptides (arrows) purified from a complex peptide mix. Panel Cshows the Control peptide (MW=1870) with one phosphothreonine and onephosphotyrosine residue after treatment with strong base (−98 amu) andmethylamine. Results show elimination of a single phosphate only (−98amu from threonine) with no subsequent addition of methylamine (+32amu), confirming a single phosphothreonine residue. Phosphotyrosine isdetermined by a lack of modification under these elimination conditions.

FIG. 7: Shows the detection of a phosphoprotein (β-casein) on a HydroGelmicroarray (Perkin Elmer, Foster City, Calif.) when the microarray wasincubated with a binding solution comprising Compound 2 and galliumchloride, see Example 18. The protein was loaded in a two-fold dilutionseries from 166 pg-0.324 pg on the microarray. The results show thedetection of 0.65 pg of a pentaphosphorylated protein on a HydroGelmicroarray.

FIG. 8: Shows the detection of a phosphopeptide (pDISP) on a HydroGelmicroarray (Perkin Elmer) when the microarray was incubated with abinding solution comprising Compound 2 and gallium chloride, see Example19. The peptide was loaded in a two-fold dilution series from 12 pg-0.18pg on the microarray. The results demonstrate that as little as 300 fgof a monophosphorylated peptide can be detected on a HydroGelmicroarray.

FIG. 9: Shows the detection of protein kinase activity (9A; CaMPKII) and(9B; Abl tyrosine kinase) by the detection of peptides that werephosphorylated on a HydroGel microarray (Perkin Elmer) that wasincubated with a binding solution comprising Compound 2 and galliumchloride, see Examples 20 and 21. The peptide glycogen synthase 1-10 wasdetected to demonstrate the kinase activity of CaMPKII and the peptideAbl was detected to demonstrate the kinase activity of Abl tyrosinekinase.

FIG. 10: Shows the detection of a (10A) phosphoprotein and (10B)phosphopeptide in solution by comparison of the polarization values withbinding solution alone and binding solution with phosphorylated andnon-phosphorylated protein or peptide, ovalbumin and deltasleep-inducing peptide, see Example 14. The binding solution alone andbinding solution in the presence of non-phosphorylated protein orpeptide demonstrates very similar fluorescence polarization andanisotropies. However, in the presence of the phosphoprotein orphosphopeptide, there is a significant increase in the fluorescencepolarization values. This result demonstrates selective binding of thephosphoprotein and phosphopeptide to the Compound 2-Ga³⁺ complex insolution but not to the non-phosphorylated protein or peptide.

FIG. 11: Shows the ratiometric analysis of proteins labeled with abinding solution of the present invention and the SYPRO® Ruby proteingel stain, demonstrating that non-specific staining and low-abundancephosphoproteins can be distinguished from non-phosphorylated proteins,see Example 22. A protein mixture containing phosphorylated andnon-phosphorylated proteins was separated on a polyacrylamide gel andthe phosphoproteins were detected with a binding solution comprisingCompound 2 and gallium chloride. All the proteins were detected when thegel was post-stained with SYPRO® Ruby protein gel stain. FIG. 11A) showsthe ratio of fluorescence intensities for ternary complexes ofphosphorylated proteins compared to total proteins. FIG. 11B) shows thefluorescence intensities of the phosphorylated protein complexes andtotal proteins plotted against the protein concentration, resulting in aconstant Y-intercept value. FIG. 11C) shows the ratio of the Y-interceptvalues when plotted against the protein concentration resulting inphosphoproteins with a ratio value 50-100 times greater than that of thenon-phosphorylated proteins.

FIG. 12: Shows the ability to detect phosphorylated kinase substrate insolution when the kinase substrate is conjugated to either Oregon Greendye label or Alexa Fluor 488 dye label using Compound 2 as thephosphate-binding compound wherein the dye labels on the substrate arequenched when a ternary complex is formed with Compound 2 and GaCl₃.FIG. 12A, is pp 60-Oregon Green 488 dye (OG); B, p-Abl-Oregon Green dye;C, pp 60-Alexa Fluor 488 dye (A488); D, pStat3-Oregon Green dye labelwherein the circles represent samples without GaCl₃ addition, and thesquares represent samples with GaCl₃ addition.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention provides phosphate-binding compounds, aphosphate-binding solution and methods for selectively detecting and/orisolating phosphorylated target molecules. The phosphate-bindingcompounds of the present invention, when present in the bindingsolution, selectively bind to phosphorylated target molecules and permitdetection and/or isolation of the target molecules. The binding solutioncomprises three critical components for binding phosphorylated targetmolecules: 1) phosphate-binding compounds; 2) a salt comprising a metalion and 3) an acid. The phosphate-binding compounds comprise a metalchelating moiety that is capable of binding the metal ion. Typically thephosphate-binding compounds are represent, but not limited, by theformula (A)m(L)n(B) wherein A is a chemical moiety, L is a linker, B isa metal-chelating moiety, m is an integer from 1 to 4 and n is aninteger from 0 to 4.

The binding solution typically includes a buffering agent to maintainthe acidic pH, which is ideally about pH 3 to about pH 6, and an organicsolvent, wherein the use and solvent depends on the application and willbe discussed below. The ternary complex that comprises thephosphate-binding compound, metal ion and phosphorylated target moleculeis stable in an acidic environment but when the pH approaches neutral(pH 7) or basic (pH>7.0) the complex becomes increasingly unstable.

The binding solution is typically used to noncovalently attach aphosphate-binding compound, of the present invention to exposedphosphate groups on phosphorylated target molecules, wherein thephosphate-binding compound typically comprises a label. Alternatively,the binding solution is used to covalently attached a phosphate-bindingcompound to a phosphorylated target molecule wherein the presentphosphate-binding compound comprises a reactive group that will form acovalent bond when brought within proximity to the phosphorylated targetmolecule. Because this is a highly directed covalent attachment, thereactive group is typically a photoactivatable group. These bound targetmolecules can be subsequently detected using one of the detectionmethods described herein or isolated by a number of methods describedbelow. The metal ions of the binding solution simultaneously haveaffinity for both phosphate groups and the metal-chelating moiety of thephosphate-binding compounds of the invention when in an acidicenvironment.

Thus, a method of the present invention for the binding ofphosphorylated target molecules by a phosphate-binding compoundcomprises the following steps:

-   -   i) contacting the sample with a binding solution, and;    -   ii) incubating the sample and the binding solution for        sufficient time to allow said compound to associate with said        phosphorylated target molecules, whereby said phosphorylated        target molecule is bound.

The methods of the present invention can be used in unlimited assayformats, provided that there is sufficient contact between the sampleand the binding solution. Therefore, this method is intended to cover anunlimited number of assays, in any format, wherein the binding solutionof the present invention has contact with an exposed phosphate group ona target molecule, regardless of the intent of the assay. Thus, themethods of the present invention contemplate, without limit, theidentification of phosphorylated target molecules, identification ofdephosphorylated molecules, identification of enzymes responsible forphosphorylation or dephosphorylation, directly or indirectly,identification of molecules that interact with phosphorylated targetmolecules and isolation of phosphorylated target molecules. Detectionincludes—where practical—quantitation, discrimination and subsequentanalysis and identification of the phosphorylated target molecules, withthe use of standards and controls, as appropriate.

DEFINITIONS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” includes plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a phosphorylated protein”includes a plurality of proteins and reference to “a compound” includesa plurality of compounds and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the invention as described herein.

The term “affinity” as used herein refers to the strength of the bindinginteraction of two molecules, such as a metal-chelating compound and ametal ion.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds of the invention may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and an acyl radical onat least one terminus of the alkane radical. The “acyl radical” is thegroup derived from a carboxylic acid by removing the —OH moietytherefrom.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”.

Exemplary alkyl groups of use in the present invention contain betweenabout one and about twenty-five carbon atoms (e.g. methyl, ethyl and thelike). Straight, branched or cyclic hydrocarbon chains having eight orfewer carbon atoms will also be referred to herein as “lower alkyl”. Inaddition, the term “alkyl” as used herein further includes one or moresubstitutions at one or more carbon atoms of the hydrocarbon chainfragment.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P and S, and wherein the nitrogen,phosphorous and sulfur atoms are optionally oxidized, and the nitrogenheteroatom is optionally be quaternized. The heteroatom(s) O, N, P, Sand Si may be placed at any interior position of the heteroalkyl groupor at the position at which the alkyl group is attached to the remainderof the molecule. Examples include, but are not limited to,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may beconsecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (preferablyfrom 1 to 3 rings), which are fused together or linked covalently.

The term “heteroaryl” as used herein refers to an aryl group as definedabove in which one or more carbon atoms have been replaced by anon-carbon atom, especially nitrogen, oxygen, or sulfur. For example,but not as a limitation, such groups include furyl, tetrahydrofuryl,pyrrolyl, pyrrolidinyl, thienyl, tetrahydrothienyl, oxazolyl,isoxazolyl, triazolyl, thiazolyl, isothiazolyl, pyrazolyl,pyrazolidinyl, oxadiazolyl, thiadiazolyl, imidazolyl, imidazolinyl,pyridyl, pyridazyl, triazinyl, piperidinyl, morpholinyl,thiomorpholinyl, pyrazinyl, piperainyl, pyrimidinyl, naphthyridinyl,benzofuranyl, benzothienyl, indolyl, indolinyl, indolizinyl, indazolyl,quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazolyl,acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, purinyl,benzimidazolyl and benzthiazolyl and their aromatic ring-fused analogs.Many fluorophores are comprised of heteroaryl groups and include,without limitations, xanthenes, oxazines, benzazolium derivatives(including cyanines and carbocyanines), borapolyazaindacenes,benzofurans, indoles and quinazolones.

The above heterocyclic groups may further include one or moresubstituents at one or more carbon and/or non-carbon atoms of theheteroaryl group, e.g., alkyl; aryl; heterocycle; halogen; nitro; cyano;hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl- or arylthio;amino, alkyl-, aryl-, dialkyl-, diaryl-, or arylalkylamino;aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl;carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- orarylsulfonyl; hydroximinyl, or aryl- or alkoximinyl. In addition, two ormore alkyl substituents may be combined to form fused heterocycle-alkylring systems. Substituents including heterocyclic groups (e.g.,heteroaryloxy, and heteroaralkylthio) are defined by analogy to theabove-described terms.

The term “heterocycloalkyl” as used herein refers to a heterocycle groupthat is joined to a parent structure by one or more alkyl groups asdescribed above, e.g., 2-piperidylmethyl, and the like. The term“heterocycloalkyl” refers to a heteroaryl group that is joined to aparent structure by one or more alkyl groups as described above, e.g.,2-thienylmethyl, and the like.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2 m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″ R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P) and silicon (Si).

The term “amino” or “amine group” refers to the group —NR′R″ (or NRR′R″)where R, R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl. Asubstituted amine being an amine group wherein R′ or R″ is other thanhydrogen. In a primary amino group, both R′ and R″ are hydrogen, whereasin a secondary amino group, either, but not both, R′ or R″ is hydrogen.In addition, the terms “amine” and “amino” can include protonated andquaternized versions of nitrogen, comprising the group —NRR′R″ and itsbiologically compatible anionic counterions.

The term “attachment site” as used herein refers to a site on a moietyor a molecule, e.g. a quencher, a fluorescent dye, an avidin, or anantibody, to which is covalently attached, or capable of beingcovalently attached, to a linker or another moiety.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “BAPTA” as used herein refers to a metal-chelating compoundthat is 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or itsanalogs, derivatives, ring-fused variants and conjugates, and allmetallic and nonmetallic salts, partial salts and hydrates thereof,including any corresponding compounds disclosed in U.S. Pat. Nos.4,603,209; 4,849,362; 5,049,673; 5,453,517; 5,459,276; 5,516,911;5,501,980; 6,162,931 and 5,773,227 (supra). When used generically,“BAPTA” refers to two benzene rings that are joined by a C₁-C₃hydrocarbon bridge terminated by oxygen atoms, including methylenedioxy(—OCH₂O—), ethylenedioxy (—OCH₂CH₂O—) or propylenedioxy (—OCH₂CH₂CH₂O—)bridging groups, where each benzene ring is optionally substituted byone or more substituents that adjust the metal ion-binding affinity,solubility, chemical reactivity, spectral properties or other physicalproperties of the compound. In a preferred embodiment of the presentinvention “BAPTA” is covalently attached to a chemical moiety A that, incombination with an appropriate trivalent metal ion and an acid, permitsdetection or isolation of phosphorylated target molecules as a ternarycomplex. BAPTA derivatives additionally include compounds in which thebenzene rings of the BAPTA structure are substituted by or fused toadditional aromatic, or heteroaromatic rings.

The term “biotin” as used herein refers to any biotin derivative,including without limitation, substituted and unsubstituted biotin, andanalogs and derivatives thereof, as well as substituted andunsubstituted derivatives of caproylamidobiotin, biocytin,desthiobiotin, desthiobiocytin, iminobiotin, and biotin sulfone.

The term “biotin-binding protein” as used herein refers to any proteinthat binds selectively to biotin, including without limitation,antibodies to biotin, substituted or unsubstituted avidin, and analogsand derivatives thereof, as well as substituted and unsubstitutedderivatives of antibodies, streptavidin, ferritin avidin, nitroavidin,nitrostreptavidin, Neutravidin™ avidin (a de-glycosylated modifiedavidin having an isoelectric point near neutral) and their dye-,enzyme-, or polymer-modified variants and immobilized forms of thebiotin-binding proteins.

The term “buffer” as used herein refers to a system that acts tominimize the change in acidity or basicity of the solution againstaddition or depletion of chemical substances.

The term “carbonyl” as used herein refers to the functional group—(C═O)—. However, it will be appreciated that this group may be replacedwith other well-known groups that have similar electronic and/or stericcharacter, such as thiocarbonyl (—(C═S)—); sulfinyl (—S(O)—); sulfonyl(—SO₂)—), phosphonyl (—PO₂—).

The term “carboxy” or “carboxyl” refers to the group —R′(COOR¹³) whereR′ is alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroaryl, or substituted heteroaryl. R¹³ ishydrogen or a salt.

The term “chemical moiety A” or “chemical moiety” as used herein refersto the moiety that is covalently attached to the metal chelatingcompound according the Formula (A)m(L)n(B) to form a phosphate-bindingcompound of the present invention. The “chemical moiety A” includes alabel, as defined below, that is a detectable moiety used to facilitatedetection and isolation of phosphorylated target molecules. The term“chemical moiety A” is a natural or syntheic moiety that is typically alabel but can also be, without limitation, a reactive group, typically aphotoactivatable group or an amine reactive group such as succinimidylester that functions to covalently bind a polymer including agarose,acrylamide, microparticles, a protein such as an antibody or an antigen,a phosphate target molecule and a ligand, including those well known toone skilled in the art, to the phosphate-binding compounds of thepresent invention. In addition, the chemical moiety A can also be ametal-chelating moiety of the present invention, typically if a metalchelating moiety is attached to a phosphate-binding compound of thepresent invention it will be through a reactive group (conjugationreaction) however the metal chelating moiety could be covalentlyattached wherein a reactive group was not used and is connected by alinker to the phosphate-binding compound of the present invention.

The term “complex” as used herein refers to the association of two ormore molecules, usually by non-covalent bonding, e.g., with a metalion-chelator and a metal ion complexed with (i.e., noncovalently boundto) a protein or, for instance, of an antibody and antigen, enzyme andenzyme substrate, ligand and receptor (e.g. biotin and avidin), nucleicacid and its complementary strand, a protein with another protein orwith a nucleic acid having affinity for the first protein, and the like.

The term “detectable response” as used herein refers to an occurrenceof, or a change in, a signal that is directly or indirectly detectableeither by observation or by instrumentation. Typically, the detectableresponse is an optical response resulting in a change in the wavelengthdistribution patterns or intensity of absorbance or fluorescence or achange in light scatter, fluorescence lifetime, fluorescencepolarization, or a combination of these parameters. Alternatively, thedetectable response is an occurrence of a signal wherein the dye isinherently fluorescent and does not produce a change in signal uponbinding to a metal ion or phosphorylated target molecule. Alternatively,the detectable response is the result of a signal, such as color,fluorescence, radioactivity or another physical property of thedetectable label becoming spatially localized in a subset of a samplesuch as in a gel, on a blot, or an array, in a well of a micoplate, in amicrofluidic chamber, or on a microparticle as the result of formationof a ternary complex of the invention that comprises a phosphorylatedtarget molecule.

The term “directly detectable” as used herein refers to the presence ofa detectable label or the signal generated from a detectable label thatis immediately detectable by observation, instrumentation, or filmwithout requiring chemical modifications or additional substances. Forexample, a fluorophore produces a directly detectable response.

The term “DTPA” as used herein refers to a metal chelating moietydiethylenetriamine pentaacetic acid or derivatives thereof and anycorresponding moieties disclosed in U.S. Pat. Nos. 4,978,763 and4,647,447. DTPA is represented by the formula(CH₂CO₂R¹³)_(Z)N[(CH₂)_(S)N(CH₂CO₂R¹³)]_(T)(CH₂)_(S)N(CH₂CO₂R¹³)_(Z)wherein the linker is attached to a methine carbon or nitrogen atom andZ is 1 or 2, S is 1 to 5, T is 0 to 4 and R¹³ is hydrogen or a salt.

The term “dye” as used herein refers to a compound that emits light toproduce an observable detectable signal. “Dye” includes fluorescent andnonfluorescent compounds that include without limitations pigments,fluorophores, chemiluminescent compounds, luminescent compounds andchromophores. The term “fluorophore” as used herein refers to acomposition that is inherently fluorescent or demonstrates a change influorescence upon binding to a biological compound or metal ion, ormetabolism by an enzyme, i.e., fluorogenic. Fluorophores may besubstituted to alter the solubility, spectral properties or physicalproperties of the fluorophore. Fluorophores of the present invention arenot sulfonated. Numerous fluorophores are known to those skilled in theart and include, but are not limited to benzofurans, quinolines,quinazolinones, indoles, benzazoles, borapolyazaindacenes and xanthenes,with the latter including fluoresceins, rhodamines and rhodols as wellas other fluorophores described in RICHARD P. HAUGLAND, MOLECULAR PROBESHANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (9^(th) edition,including the CD-ROM, September 2002).

The term “energy transfer” as used herein refers to the process by whichthe excited state energy of an excited group, e.g. fluorescent reporterdye, is conveyed through space or through bonds to another group, e.g. aquencher moiety or fluorescer, which may attenuate (quench) or otherwisedissipate or transfer the energy to another reporter group or emit theenergy at a longer wavelength. Energy transfer typically occurs throughfluorescence resonance energy transfer (FRET).

The term “energy transfer pair” as used herein refers to any twomoieties that participate in energy transfer. Typically, one of themoieties acts as a fluorescent reporter, i.e. donor, and the other actsas an acceptor, which may be a quenching compound or a compound thatabsorbs and re-emits energy in the form of a fluorescent signal(“Fluorescence resonance energy transfer.” Selvin P. (1995) MethodsEnzymol 246:300-334; dos Remedios C. G. (1995) J. Struct. Biol.115:175-185; “Resonance energy transfer: methods and applications.” WuP. and Brand L. (1994) Anal Biochem 218:1-13). Fluorescence resonanceenergy transfer (FRET) is a distance-dependent interaction between twomoieties in which excitation energy, i.e. light, is transferred from adonor to an acceptor without emission of a photon. The acceptor may befluorescent and emit the transferred energy at a longer wavelength, orit may be non-fluorescent and serve to diminish the detectablefluorescence of the reporter molecule (quenching). FRET may be either anintermolecular or intramolecular event, and is dependent on the inversesixth power of the separation of the donor and acceptor, making ituseful over distances comparable with the dimensions of biologicalmacromolecules. Thus, the spectral properties of the energy transferpair as a whole change in some measurable way if the distance betweenthe moieties is altered by some critical amount. Self-quenching probesincorporating fluorescent donor-non-fluorescent acceptor combinationshave been developed primarily for detection of proteolysis (Matayoshi,(1990) Science 247:954-958) and nucleic acid hybridization (“Detectionof Energy Transfer and Fluorescence Quenching” Morrison, L., inNonisotopic DNA Probe Techniques, L. Kricka, Ed., Academic Press, SanDiego, (1992) pp. 311-352; Tyagi S. (1998) Nat. Biotechnol. 16:49-53;Tyagi S. (1996) Nat. Biotechnol 14:303-308). In most applications, thedonor and acceptor dyes are different, in which case FRET can bedetected by the appearance of sensitized fluorescence of the acceptor orby quenching of donor fluorescence.

The term “enzyme” as used herein refers to a protein molecule producedby living organisms, or through chemical modification of a naturalprotein molecule, that catalyzes a chemical reaction of other substanceswithout itself being destroyed or altered upon completion of thereactions. Examples of other substances, include, but are not limited tochemiluminescent, chromogenic and fluorogenic substances orprotein-based substrates.

The term “IDA” as used herein refers to imidodiacetic acid metalchelating moieties having the formula —N(CH₂CO₂R¹³)₂ wherein R¹³ ishydrogen or a salt and the linker is attached to the nitrogen atomprovided that the linker is not a single covalent bond attached to anaromatic ring of a fluorophore.

The term “isolated” as used herein with reference to the subjectpeptides, proteins and protein complexes, refers to a preparation of apeptide, protein or protein ternary complex that is essentially freefrom contaminating nonphosphorylated peptides, proteins or otherassociated target molecules that normally would be present inassociation with the peptide, protein or complex, e.g., in a cellularmixture or milieu in which the protein or complex is found endogenously.In addition, in some embodiments, “isolated” also refers to the furtherseparation from reagents of the invention used to isolate the peptide,protein or complex from cellular mixture. Thus, an isolated protein orprotein complex is separated (isolated) from other components of thesample and optionally from the phosphate-binding compounds of theinvention (including polymeric matrices) that normally would“contaminate” or interfere with the study or further processing of thecomplex in isolation, such as by mass spectrometry. The term “isolated”can also refer to phosphorylated target molecules that are spatially ortemporally separated from each other such as by different physicallocations on a gel or array or by having different passage times througha detector such as in a column or capillary.

The term “kit” as used herein refers to a packaged set of relatedcomponents, typically one or more compounds or compositions, optionallycomprising buffers, separation media, standards, software and othercomponents.

The term “label” as used herein refers to a detectable moiety that isused to facilitate detection and isolation of phosphorylated targetmolecules in combination with the metal-chelating moieties of thepresent invention. Illustrative labels include labels that can bedirectly observed or measured or indirectly observed or measured such asfluorophores, radioactive and enzyme reporter labels (Patton, W., et al,J. Chromatography B: Biomedical Applications (2002) 771:3-31; Patton,W., et al, Electrophoresis (2000) 21:1123-1144). Such labels include,but are not limited to, radiolabels that can be measured withradiation-counting devices; pigments, dyes or other chromogens that canbe visually observed or measured with a spectrophotometer; spin labelsthat can be measured with a spin label analyzer; and fluorescent labels(fluorophores), where the output signal is generated by the excitationof a suitable molecular adduct and that can be visualized by excitationwith light that is absorbed by the dye or can be measured with standardfluorometers or imaging systems, for example or metal particles, e.g.gold or silver particles or metallic bar codes that can be detected bytheir optical or light-scattering properties. The label can be achemiluminescent substance, where the output signal is generated bychemical modification of the signal compound; a metal-containingsubstance; or an enzyme, where there occurs an enzyme-dependentsecondary generation of signal, such as the formation of a coloredproduct from a colorless substrate. The term label can also refer to a“tag”, hapten or other ligand that can bind selectively to a labeledmolecule such that the labeled molecule, when added subsequently, isused to generate a detectable signal. For example, one can use biotin asa tag and then use an avidin or streptavidin conjugate of horseradishperoxidase (HRP) to bind to the tag, and then use a chromogenicsubstrate (e.g., tetramethylbenzidine) or a fluorogenic substrate suchas Amplex® Red reagent (Molecular Probes, Inc.) to detect the presenceof HRP. Numerous labels and tags and methods for their selectivedetection are known by those of skill in the art and include, but arenot limited to, particles, fluorophores, haptens, enzymes and theirchromogenic, fluorogenic and chemiluminescent substrates and otherlabels that are described in the MOLECULAR PROBES HANDBOOK, supra. Inaddition, present labels can be substituted with substitutents thatalter the ion-binding affinity of the phosphate binding compound,solubility, chemical reactivity, spectral properties or other physicalproperties of the label provided that the label is not sulfonated.

The term “Linker” or “L”, as used herein, refers to a single covalentbond or a series of stable covalent bonds incorporating 1-30 nonhydrogenatoms selected from the group consisting of C, N, O, S and P thatcovalently attach the phosphate-binding compounds to another moiety suchas a chemically reactive group or a phosphorylated target molecule.Exemplary linking members include a moiety that includes —C(O)NH—,—C(O)O—, —NH—, —S—, —O—, and the like. A “cleavable linker” is a linkerthat has one or more cleavable groups that may be broken by the resultof a reaction or condition. The term “cleavable group” refers to amoiety that allows for release of a portion, e.g., a label orphosphorylated target molecule, of a conjugate from the remainder of theconjugate by cleaving a bond linking the released moiety to theremainder of the conjugate. Such cleavage is either chemical in nature,or enzymatically mediated. Exemplary enzymatically cleavable groupsinclude natural amino acids or peptide sequences that end with a naturalamino acid.

In addition to enzymatically cleavable groups, it is within the scope ofthe present invention to include one or more sites that are cleaved bythe action of an agent other than an enzyme. Exemplary non-enzymaticcleavage agents include, but are not limited to, acids, bases, light(e.g., nitrobenzyl derivatives, phenacyl groups, benzoin esters), andheat. Many cleaveable groups are known in the art. See, for example,Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al.,J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol.,124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155: 141-147(1986); Park et al., J. Biol. Chem., 261: 205-210 (1986); Browning etal., J. Immunol., 143: 1859-1867 (1989). Moreover a broad range ofcleavable, bifunctional (both homo- and hetero-bifunctional) spacer armsare commercially available.

An exemplary cleavable group, an ester, is cleavable group that may becleaved by a reagent, e.g. sodium hydroxide, resulting in acarboxylate-containing fragment and a hydroxyl-containing product.

The linker can be used to attach the compound to another component of aconjugate, such as a targeting moiety (e.g., antibody, ligand,non-covalent protein-binding group, etc.), an analyte, a biomolecule, adrug and the like.

The term “metal chelator” or “metal-chelating moiety” as used hereinrefers to a chemical moiety that combines with a metal ion to form achelate ring structure. For the purposes of the present invention themetal chelator has affinity for a metal ion that has simultaneousaffinity for the metal chelator and a phosphate target molecule in amoderately acidic environment. Examples of metal-chelating moietiesinclude, but are not limited to, BAPTA, IDA, DTPA and phenanthroline.The metal chelators are optionally substituted by substituents thatadjust the ion-binding affinity, solubility, chemical reactivity,spectral properties or other physical properties of the compoundprovided that the metal chelator is not sulfonated.

The term “metal ion” as used herein refers to any trivalent metal ionthat has simultaneous affinity for a phosphate group of a targetmolecule and a metal-chelating compound of the invention at pH 3 to 6and that can be used to form a ternary complex of the phosphate-bindingcompound and the phosphorylated target molecule. Such metal ionsinclude, without limitation, Al³⁺, Fe³⁺ and Ga³⁺. For purposes of thepresent invention, the metal ion must have simultaneous affinity forboth the metal-chelating moiety and phosphate groups of the targetmolecule and, as such, confers affinity to the metal-chelating moietyfor the phosphate groups of the target molecule that would not bepresent without the metal ion.

The term “phosphate-binding compound” or “binding compound” as usedherein refers to a compound that is capable of binding a metal ionwherein the metal ion has simultaneous affinity for a phosphorylatedtarget molecule. Such phosphate-binding compounds are typicallyrepresented by, but are not limited to, the formula (A)m(L)n(B)n whereinA is a chemical moiety, L is a linker, B is metal-chelating moiety, m isan integer from 1 to 4 and n is an integer from 0 to 4. These compoundseffectively, but non-covalently, attach a label to a phosphorylatedtarget molecule when the metal-chelating moiety indirectly bindsphosphate groups on the target molecule.

The terms “phosphorylated target molecule” or “phosphate targetmolecule” as used herein refers to a molecule possessing one or morephosphate or phosphate analog moieties each attached to such molecule bya single ester bond or inorganic phosphate. Phosphate analogs include,without limitation, thiophosphate, boraphosphate, phosphoramide,H-phosphonate, alkylphosphonate, phosphorothioate, phosphorodithioateand phosphorofluoridate. Phosphorylated target molecules include, butare not limited to, phosphoproteins, phosphopeptides, phospholipids,phosphoglycans, phosphocarbohydrates, phosphoamino acids, pyrophosphateand inorganic phosphate and their thiophosphate analogs. Most knownphosphate compounds, and subsequently the phosphorylated targetmolecules, can be categorized into one of three groups; 1) individualphosphate groups (e.g., inorganic phosphate or a phosphate group (PO₃)on a protein or peptide); 2) multiple-linked phosphate group (e.g.,pyrophosphate or a nucleotide such as ATP); or 3) bridging phosphategroup (i.e., nucleic acids). For the purposes of the present invention,phosphorylated target molecules do not include molecules in the thirdgroup, e.g., DNA or RNA. Typically, phosphoproteins and phosphopeptidesare phosphorylated post-translationally on the tyrosine, serine orthreonine amino acid residues. Other phosphorylated amino acid residuesin peptides and proteins include 1-phospho-histidine,3-phospho-histidine, phospho-aspartic acid, phospho-glutamic acid andless commonly N′-phospho-lysine, N^(ω)-phospho-arginine andphospho-cysteine (Kaufmann, et al (2001) Proteomics 1: 194-199; Yan, J.,Paxker, N., Gooley, A. and Williams, K. (1998) J. Chromatograph. A 808:23-41). Thus, a phosphorylated protein or peptide typically comprises atleast one of these amino acid residues. Phosphorylated target moleculesalso include phosphorylated proteins that incorporate other non-peptideregions such as lipids or carbohydrates, e.g., lipoproteins andlipopolysaccharides. In addition, the lipid or carbohydrate residues ofthe proteins can be phosphorylated instead or in combination with thetyrosine, serine or threonine amino acid residues of the proteins andpeptides such as a phosphomannose-modified orN-acetylglucosamine-1-phosphate modified protein. Other modificationsinclude a pyridoxal phosphate Schiff base to the epsilon-amino group oflysine, and an O-pantetheine phosphorylation of serine residue. Thegamma phosphate of nucleotide triphosphates is also detectable using themethods of this invention, making photolabeled proteins and peptidesdetectable by this procedure. For the purposes of the present inventionphosphorylated target molecules include phosphorylated lipids andcarbohydrates.

The term “photoactivatable reactive group” as used herein refers to achemical moiety that becomes chemically active by exposure to anappropriate wavelength, typically a UV wavelength. Once activated thereactive group is capable of forming a covalent bond with a proximalmoiety on a biological or non-biological component. In the instant case,the phosphate-binding compounds may contain a photoactivatable groupthat can form a covalent bond with a phosphorylated target molecule whenbrought within proximity by the formation of the ternery complex andactivated by an appropriate wavelength. Photoactivatable groups include,but are not limited to, benzophenones, aryl azides and diazirines.

The terms “protein” and “polypeptide” are used herein in a generic senseto include polymers of amino acid residues of any length. The term“peptide” is used herein to refer to polypeptides having less than 100amino acid residues, typically less than 15 amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The peptide or protein may be further conjugated to orcomplexed with other moieties such as dyes, haptens, radioactiveisotopes, natural and synthetic polymers (including microspheres),glass, metals and metallic particles, proteins and nucleic acids.

The term “reactive group” as used herein refers to a group that iscapable of reacting with another chemical group to form a covalent bond,i.e. is covalently reactive under suitable reaction conditions, andgenerally represents a point of attachment for another substance. Thereactive group is a moiety, such as a photoactivatable group, carboxylicacid or succinimidyl ester, on the compounds of the present inventionthat is capable of chemically reacting with a functional group on adifferent compound to form a covalent linkage resulting in aphosphate-binding labeled component. Reactive groups generally includenucleophiles, electrophiles and photoactivatable groups.

Exemplary reactive groups include, but not limited to, olefins,acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes,ketones, carboxylic acids, esters, amides, cyanates, isocyanates,thiocyanates, isothiocyanates, amines, hydrazines, hydrazones,hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides,disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids,acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles,amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamicacids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines,ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates,imines, azides, azo compounds, azoxy compounds, and nitroso compounds.Reactive functional groups also include those used to preparebioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and thelike. Methods to prepare each of these functional groups are well knownin the art and their application to or modification for a particularpurpose is within the ability of one of skill in the art (see, forexample, Sandler and Karo, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS,Academic Press, San Diego, 1989).

The term “sample” as used herein refers to any material that may containphosphorylated target molecules, natural or synthetic, as defined above,or contains components that directly interact with phosphate orphosphorylated target molecules, such as enzymes. Typically, the samplecomprises purified or semi-purified phosphorylated target molecules andendogenous host cell proteins. The phosphorylated target molecules canbe made synthetically or obtained in a purified or semi-purified formfrom cells (eukaryotic and prokaryotic, without limitation) cellextracts, cell homogenates, subcellular components as natural orrecombinant molecules. Alternatively, phosphorylated target moleculescan be obtained from tissue homogenate, bodily and other biologicalfluids, or synthesized proteins, all of which comprise a sample in thepresent invention. The sample may be in an aqueous or mostly aqueoussolution, a viable cell culture or immobilized on a solid or semi solidsurface such as a polymer gel, a membrane, a microparticle, an opticalfiber or on a microarray. In addition “sample” as use herein also refersto substrates for kinases or phosphatases or molecules that bindphosphorylated target molecules that may or may not be phosphorylated.In this way the sample comprises components that interact with phosphateand phosphorylated target molecules, particularly including antibodiesto either the phosphorylated target molecules or to other regions of thetarget molecule or, for instance, complexes of biotinylated targetmolecules with an avidin derivative.

The term “ternary complex” as used herein refers to a composition thatsimultaneously comprises a phosphate-binding compound, a trivalent metalion of the present invention and a phosphate target molecule, whereinthe metal ions simultaneously have affinity for both the metal-chelatingmoiety of the compound and the phosphate group on the molecule, andwherein the metal ion forms a bridge between the two molecules. Unlesslimited by the context of their use, the terms “binding” and “complexformation” in this invention mean the process of formation of thisternary complex.

The Phosphate-Binding Compounds

In general, for ease of understanding the present invention, thephosphate-binding compounds and components of the binding solution willfirst be described in detail, followed by the many and varied methods inwhich the phosphate-binding compounds and metal ions find uses, which isfollowed by exemplified methods of use and synthesis of certain novelcompounds that are particularly advantageous for use with the methods ofthe present invention.

The phosphate-binding compounds of the present invention arecharacterized as being capable of binding to a trivalent metal ion thathas simultaneous affinity for a phosphorylated target molecule to form aternary complex. Thus, any metal chelating moiety that is capable ofbinding a trivalent metal ion wherein the metal ion has affinity for aphosphorylated target molecule is contemplated and considered part ofthe present invention. For detection, isolation and quantificationpurposes the metal chelating moiety is typically covalently attached toa reactive group or a label that typically a dye, a hapten or an enzyme,wherein the reactive group or label are collectively defined herein as“chemical moiety A”.

Thus, the present phosphate-binding compounds are typically representedby, but not limited to, the formula (A)(B) wherein A is the chemicalmoiety and B is the metal chelating moiety. More typically, the presentphosphate-binding compounds are represented by the formula (A)m(L)n(B),wherein A is a chemical moiety and L is a linker that covalentlyattaches the chemical moiety to the metal-chelating moiety (B) and m andn are individually integers between 0 and 4. The metal-chelating moietyis dictated by metal ions that have affinity for phosphate and phosphateanalog groups; such ions include, but are not limited to, Ga³⁺, Fe³⁺ andAl³⁺. It was found that for purposes of the present invention trivalentgallium ions when in a moderately acidic environment, e.g. between aboutpH 3 and about pH 6, have affinity for phosphate groups on targetmolecules and certain chelating groups such as BAPTA, IDA, DTPA andphenanthroline; BAPTA chelating moieties are the most preferred.

Metal Chelating Moieties

The metal-chelating moieties are moieties characterized as being capableof simultaneously binding metal ions that have affinity for exposedphosphate groups on target molecules, wherein a ternary complex isformed between the metal-chelating moiety, the metal ion and thephosphorylated target molecule. Metal ions that have been found to bindphosphate groups include, without limitation, trivalent gallium, ironand aluminum. Metal-chelating moieties that bind these ions, undercertain conditions, include, without limitation, BAPTA, IDA, DTPA andphenanthrolines. Thus, the metal-chelating moieties must 1) bind metalions that have affinity for phosphate groups, 2) not interfere with thebinding of the metal ion for the phosphate groups and 3) maintain astable ternary complex. Exemplary metal-chelating moieties that fitthese three criteria include BAPTA, IDA, DTPA and phenanthrolines.

BAPTA, as used herein, refers to analogs, including fluorescent andnonfluorescent derivatives, of the metal-chelating moiety(1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) and saltsthereof including any corresponding compounds disclosed in U.S. Pat.Nos. 4,603,209; 4,849,362; 5,049,673; 5,453,517; 5,459,276; 5,516,911;5,501,980; and 5,773,227. These BAPTA-based metal-chelating moieties arewell known to those skilled in the art, primarily as calcium indicatorsdue to their ability to bind divalent calcium ions under physiologicalconditions, i.e. a pH of about 7 and free calcium ion concentrationsnear the micromolar and submicromolar range. As calcium indicators thesecompounds are typically used in live cells wherein the indicators arederivatized on a carboxylic group to comprise at least one lipophilicgroup or specifically an acetoxymethyl (AM) ester group, wherein AMester is represented as —CH₂OCOCH₃, to produce cell permeant derivativesof the indicators.

However, we found that calcium is a totally ineffective metal ion forpractice of the methods of the present invention to detectphosphorylated target molecules described in this invention with theseindicators.

For the sake of clarity the following structure represents preferredpresent BAPTA metal-chelating moieties having Formula IV:

Preferably the two rings are linked by a hydrocarbon bridge between twooxygen atoms in which p is 0, 1 or 2 and the ring substituents (R¹-R⁸)are selected independently from the group consisting of hydrogen,halogen, hydroxyl, alkoxy, alicyclic, heteroalicyclic, alkyl, aryl,amino, aldehyde, carboxyl, nitro, cyano, thioether, sulfinyl and linker(L). Alternatively, two adjacent ring substituents in combinationconstitute a cyclic substituent that is cycloalkyl, cycloheteroalkyl,aryl, fused aryl, heteroaryl or fused heteroaryl. Preferably, the BAPTAmetal-chelating moieties have at least two substituents that are nothydrogen, a most preferred BAPTA metal-chelating moiety is substitutedby a fluorine atom as one of the substituents, most preferablysubstituted at the R⁶ or R³ position (e.g., Compounds 1, 2, 5, 7, 8 and12). Typically the linker attaching the chemical moiety to the BAPTA isat the R³ or R⁶ position. Equally preferred are BAPTA metal-chelatingmoieties that comprise a carbonyl group as a substituent, preferably atthe R⁷ position, e.g., Compounds 9 and 12. Without being bound by aparticular theory, it appears that an electron withdrawing group such asfluorine or carbonyl substituted at the R³, R⁴, R⁶ or R⁷ positionresults in BAPTA chelating moieties that are particularly advantageousfor chelating trivalent gallium ions that then also allows for thesimultaneous interaction of the chelated gallium ion with an exposedphosphate group on the phosphorylated target molecules, resulting in astable ternary complex.

The bridge substituents R⁹, R¹⁰, R¹¹ and R¹², are independently selectedfrom the group consisting of hydrogen, lower alkyl, or adjacentsubstituents R⁹ and R¹⁰, taken in combination, constitute a 5-memberedor 6-membered alicyclic or heterocyclic ring. R¹⁵, R¹⁶, R¹⁷ and R¹⁸ areindependently H or lower alkyl; preferably R¹⁵, R¹⁶, R¹⁷ and R¹⁸ are allhydrogen. R¹³ and R¹⁴ are independently hydrogen or a salt.

It is understood that the chemical moieties of the present invention areattached to the BAPTA metal-chelating moiety by a linker at any ofR¹-R¹² or alternatively the dye label comprises one of the aromaticrings of the metal-chelating moieties wherein no linker is present.Therefore, two adjacent substituents of R¹-R¹², when taken incombination with each other, and with the aromatic ring to which theyare bound, comprise a fluorophore or chromophore label. However, aphosphate-binding compound could have more than one linker, such that adye label is attached with no linker and four other linkers are presenton the metal chelating compound to attach other labels or reactivegroups. In one aspect of the invention, two adjacent ring substituents(R¹-R⁴ or R⁵-R⁸) taken in combination form the dye label that is a fusedbenzofuran or heteroaryl- or carboxyheteroaryl-substituted benzofuranfluorophore. Where the dye label is fused to the compound of theinvention, it is preferably fused between R² and R³, or between R⁶ andR⁷.

Xanthene derivative dyes are particularly useful dyes of the presentinvention wherein, either or both of the benzene rings of the BAPTA orsubstituted BAPTA metal-binding compound is bonded to a xanthene ringthrough a single chemical bond, as in the common Ca²⁺ indicators fluo-3,fluo-4 and rhod-2 (U.S. Pat. No. 5,049,673, supra) or through theintermediacy of a phenyl or substituted phenyl spacer as in the OregonGreen® BAPTA indicators (U.S. Pat. No. 6,162,931, supra). The xanthenerings are typically bonded to the BAPTA at positions para to thenitrogen functions of the BAPTA. Particularly preferred arexanthene-containing BAPTA derivatives whose fluorophore is a rhodamineor a halogenated fluorescein. Particularly preferred are fluorescentBAPTA derivatives in which the 5-position of the BAPTA chelator issubstituted by F, including rhod-5F and fluo-5F.

DTPA, as used herein, refers to diethylenetriamine pentaacetic acidchelating moieties and derivatives thereof, including any correspondingcompounds disclosed in U.S. Pat. Nos. 4,978,763 and 4,647,447. DTPAmetal-chelating moieties are represented by Formula V comprising(CH₂CO₂R¹³)_(Z)N[(CH₂)_(S)N(CH₂CO₂R¹³)]_(T)(CH₂)_(S)N(CH₂CO₂R¹³)_(Z),wherein a linker is attached to a methine carbon or nitrogen atom, Z is1 or 2, S is 1 to 5, T is 0-4 and R¹³ is independently a hydrogen or asalt.

IDA, as used herein, refers to iminodiacetic acid compounds andderivatives thereof and is represented by Formula VI comprising-(L)-N(CH₂CO₂R¹³)₂ wherein R¹³ is independently a hydrogen or a salt andprovided that said linker is not a single covalent bond. The IDAmetal-chelating moieties must be attached by a linker to a chemicalmoiety wherein the linker comprises at least one nonhydrogen atom.Without wishing to be bound by a theory, it appears that the linkerincreases the stability of the ternary complex and possibly tunes theaffinity of the metal-chelating moiety for a metal ion of the presentinvention.

In addition to the above mentioned specific metal chelating moieties wehave also found that phenanthroline based chelators also form ternarycomplex with metal ions and phosphate target molecules in a moderatelyacidic environment. Phenanthroline, as used herein, refers to1,10-phenanthroline compounds and derivatives thereof and is representedby the structure

Any of the aromatic carbon atoms may be substituted with substituentswell known to one skilled in the art, including those substituentsdisclosed in U.S. Pat. No. 6,316,267, supra. Alternatively, a linker canbe attached to any of the aromatic carbon atoms to covalently attach achemical moiety A to the phenanthroline moiety to form thephosphate-binding compounds of the present invention.

Labels

In certain embodiments, the present phosphate-binding compounds comprisea label that is covalently bonded to a present metal chelating moiety.The present labels are characterized as being any label known to oneskilled in the art and when the label is either covalently linked to ametal-chelating moiety or comprises part of the metal-chelating moietywherein no linker is present, forms a present phosphate-bindingcompound. Labels include, without limitation, a chromophore, afluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye(energy transfer pair), a microparticle, a polymer, a hapten, an enzymeand a radioisotope. Preferred labels include dyes, fluorescent proteins,haptens, and enzymes. The covalent linkage can be a single covalent bondor a combination of stable chemical bonds. The covalent linkage bindingthe label to the metal-chelating moiety is typically a single covalentbond, but can also be a substituted alkyl chain that incorporates 1-30nonhydrogen atoms, or a substituted cycloalkyl, selected from the groupconsisting of C, N, O, S and P.

A dye of the present invention is any chemical moiety that exhibits anabsorption maximum beyond 280 nm, that when part of a phosphate-bindingcompound retains its unique spectral properties to provide a detectablesignal. The preferred dyes are fluorophores or chemiluminescenceprecursors that are directly detectable or that upon action of anadditional reagent or reagents yield fluorescence or chemiluminescence.

Dyes of the present invention include, without limitation; a pyrene, ananthracene, a naphthalene, an acridine, a stilbene, an indole orbenzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine including anycorresponding compounds in U.S. Ser. Nos. 09/968,401 and 09/969,853 andU.S. Pat. Nos. 6,403,807 and 6,348,599), a carbocyanine (including anycorresponding compounds in U.S. Ser. No. 09/557,275 and U.S. Pat. Nos.5,486,616; 5,268,486; 5,569,587; 5,569,766; 5,627,027 and 6,048,982), acarbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, aperylene, a pyridine, a quinoline, a borapolyazaindacene (including anycorresponding compounds disclosed in U.S. Pat. Nos. 4,774,339;5,187,288; 5,248,782; 5,274,113; and 5,433,896, supra), a xanthene(including any corresponding compounds disclosed in U.S. Pat. Nos.6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343 6,221,606;6,358,684; 6,008,379; 6,111,116; 6,184,379; 6,017,712; 6,080,852;5,847,162 and U.S. Ser. No. 09/922,333) an oxazine or a benzoxazine, acarbazine (including any corresponding compounds disclosed in U.S. Pat.No. 4,810,636), a phenalenone, a coumarin (including an correspondingcompounds disclosed in U.S. Pat. Nos. 5,696,157; 5,459,276; 5,501,980and 5,830,912), a benzofuran (including any corresponding compoundsdisclosed in U.S. Pat. Nos. 4,603,209 and 4,849,362) and benzphenalenone(including any corresponding compounds disclosed in U.S. Pat. No.4,812,409) and derivatives thereof. As used herein, oxazines includeresorufins (including any corresponding compounds disclosed in U.S. Pat.No. 5,242,805), aminooxazinones, diaminooxazines, and theirbenzo-substituted analogs.

Where the dye is a xanthene, the dye is optionally a fluorescein, arhodol (including any corresponding compounds disclosed in U.S. Pat.Nos. 5,227,487 and 5,442,045), a rhodamine (including any correspondingcompounds in U.S. Pat. Nos. 5,798,276 and 5,846,737). As used herein,rhodamine and rhodol dyes include, among other derivatives, compoundsthat comprise xanthenes with saturated or unsaturated “julolidine”rings. As used herein, fluorescein includes benzo- ordibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins.Similarly, as used herein rhodol includes seminaphthorhodafluors(including any corresponding compounds disclosed in U.S. Pat. No.4,945,171).

Preferred dyes of the present invention include benzofurans, quinolines,quinazolinones, xanthenes, indoles, benzazoles and borapolyazaindacenes.Preferred xanthenes include julolidine-containing xanthenes, as well asfluoresceins, rhodols, rhodamines and rosamines. Xanthenes of thisinvention comprise both compounds substituted and unsubstituted on thecarbon atom of the central ring of the xanthene by substituentstypically found in the xanthene-based dyes such as phenyl andsubstituted-phenyl moieties. It is an important aspect of the currentinvention that none of the preferred fluorescent dyes are sulfonated.

Alternatively, the dye is a xanthene that is bound via an L that is asingle covalent bond at the 9-position of the xanthene. Preferredxanthenes include derivatives of 3H-xanthen-6-ol-3-one attached at the9-position, derivatives of 6-amino-3H-xanthen-3-one attached at the9-position, or derivatives of 6-amino-3H-xanthen-3-imine attached at the9-position.

Typically the dye contains one or more aromatic or heteroaromatic ringsthat are optionally substituted one or more times by a variety ofsubstituents, including without limitation, halogen, nitro, cyano,alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl,acyl, aryl or heteroaryl ring system, benzo, or other substituentstypically present on dyes known in the art.

In one aspect of the invention, the dye has an absorption maximum beyond480 nm. In a particularly useful embodiment, the dye absorbs at or near488 nm to 514 nm (particularly suitable for excitation by the output ofthe argon-ion laser excitation source) or near 546 nm (particularlysuitable for excitation by a mercury arc lamp). As is the case for manydyes, they can also function as both chromophores and fluorophores,resulting in compounds that simultaneously act both as colorimetric andfluorescent labels for phosphorylated target molecules. Thus, thedescribed fluorescent dyes are also the preferred chromophores of thepresent invention.

In addition to dyes, enzymes also find use as labels for thephosphate-binding compounds having the formula (A)m(L)n(B). Enzymes aredesirable labels because amplification of the detectable signal can beobtained resulting in increased assay sensitivity. The enzyme itselfdoes not produce a detectable response but functions to break down asubstrate when it is contacted by an appropriate substrate such that theconverted substrate produces a fluorescent, calorimetric or luminescentsignal. Enzymes amplify the detectable signal because one enzyme on alabeling compound can result in multiple substrate molecules beingconverted to a detectable signal. This is advantageous where there is alow quantity of phosphorylated target molecules present in the sample ora fluorophore does not exist that will give comparable or strongersignal than the enzyme. Fluorophores are most preferred because they donot require additional assay steps that can lead to an unstable ternarycomplex. The enzyme substrate is selected to yield the preferredmeasurable product, e.g. color, fluorescence or chemiluminescence. Suchsubstrates are extensively used in the art, many of which are describedin the MOLECULAR PROBES HANDBOOK, supra.

A preferred calorimetric or fluorogenic substrate and enzyme combinationuses oxidoreductases such as horseradish peroxidase (HRP) and asubstrate such as 3,3′-diaminobenzidine (DAB) or3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color(brown and red, respectively). Other calorimetric oxidoreductasesubstrates that yield detectable products include, but are not limitedto: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid and 4-chloro-1-naphthol.Fluorogenic substrates include, but are not limited to, homovanillicacid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines andreduced benzothiazines, including Amplex® Red reagent and its variants(U.S. Pat. No. 4,384,042) and reduced dihydroxanthenes, includingdihydrofluoresceins (U.S. Pat. No. 6,162,931) and dihydrorhodamines,including dihydrorhodamine 123. Peroxidase substrates that are tyramides(U.S. Pat. Nos. 5,196,306; 5,583,001 and 5,731,158) represent a uniqueclass of peroxidase substrates in that they can be intrinsicallydetectable before action of the enzyme but are “fixed in place” by theaction of a peroxidase in the process described as tyramide signalamplification (TSA). These substrates are extensively utilized to labeltargets in samples that are cells, tissues or arrays for theirsubsequent detection by microscopy, flow cytometry, optical scanning andfluorometry.

Another preferred colorimetric (and in some cases fluorogenic) substrateand enzyme combination uses a phosphatase enzyme such as an acidphosphatase or a recombinant version of such a phosphatase incombination with a colorimetric substrate such as5-bromo-4-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolylphosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenylphosphate, or o-nitrophenyl phosphate or with a fluorogenic substratesuch as 4-methylumbelliferyl phosphate,6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat.No. 5,830,912), fluorescein diphosphate, 3-O-methylfluoresceinphosphate, resorufin phosphate,9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate (DDAOphosphate), or ELF 97, ELF 39 or related phosphates (U.S. Pat. Nos.5,316,906 and 5,443,986).

Glycosidases, in particular β-galactosidase, β-glucuronidase andβ-glucosidase, are additional suitable enzymes. Appropriate colorimetricsubstrates include, but are not limited to, 5-bromo-4-chloro-3-indolylβ-D-galactopyranoside (X-gal) and similar indolyl galactosides,glucosides, and glucuronides, o-nitrophenyl β-D-galactopyranoside (ONPG)and p-nitrophenyl β-D-galactopyranoside. Preferred fluorogenicsubstrates include resorufin β-D-galactopyranoside, fluoresceindigalactoside (FDG), fluorescein diglucuronide and their structuralvariants (U.S. Pat. Nos. 5,208,148; 5,242,805; 5,362,628; 5,576,424 and5,773,236), 4-methylumbelliferyl β-D-galactopyranoside,carboxyumbelliferyl β-D-galactopyranoside and fluorinated coumarinβ-D-galactopyranosides (U.S. Pat. No. 5,830,912).

Additional enzymes include, but are not limited to, hydrolases such ascholinesterases and peptidases, oxidases such as glucose oxidase andcytochrome oxidases and reductases for which suitable substrates areknown.

Enzymes and their appropriate substrates that produce chemiluminescenceare preferred for some assays. These include, but are not limited to,natural and recombinant forms of luciferases and aequorins.Chemiluminescence-producing substrates for phosphatases, glycosidasesand oxidases such as those containing stable dioxetanes, luminol,isoluminol and acridinium esters are additionally useful. Severalchemiluminescent substrates for phosphatase enzymes are known, includingthe BOLD APB chemiluminescent substrate (Molecular Probes, Inc.).

In addition to enzymes, haptens such as biotin, digoxigenin and2,4-dinitrophenyl are also preferred labels. Biotin is useful because itcan function in an enzyme system to further amplify the detectablesignal, and it can function as a tag to be used in affinitychromatography for isolation purposes. For detection purposes, an enzymeconjugate that has affinity for biotin is used, such as avidin-HRP.Subsequently, a peroxidase substrate is added to produce a detectablesignal. For isolation purposes, a protein such as avidin that hasaffinity for biotin is conjugated to agarose beads. The biotin-labeledmetal-chelating moiety, after contacting a phosphorylated targetmolecule, is then incubated with the avidin beads, on a column, bound toa magnetic particle or in solution, to separate and/or concentrate thephosphorylated target molecules. A preferred form of biotin is thedesthiobiotin analog, which can be easily adsorbed and released fromavidin-based affinity matrices. A preferred form of avidin for someapplications is CaptAvidin biotin-binding protein (Molecular Probes),which permits facile release of biotinylated compounds.

Haptens also include, among other derivatives, hormones, naturallyoccurring and synthetic drugs, pollutants, allergens, affectormolecules, growth factors, chemokines, cytokines, lymphokines, aminoacids, peptides, chemical intermediates, nucleotides and the like.

Fluorescent or luminescent proteins also find use as labels for thephosphate-binding compounds of the present invention. Examples offluorescent proteins include green-fluorescent protein (GFP), acquorinand the phycobiliproteins and the derivatives thereof. The fluorescentproteins, especially phycobiliproteins, are particularly useful forcreating tandem dye-labeled labeling reagents or for indirect detectionof hapten-labeled labeling compounds or phosphorylated target moleculesthat are immobilized on a matrix, such as a microsphere or an array.These tandem dyes comprise a fluorescent protein and a fluorophore forthe purposes of obtaining a larger Stokes shift, wherein the emissionspectra are farther shifted from the wavelength of the fluorescentprotein's absorption spectra. This property is particularly advantageousfor detecting a low quantity of a target molecule in a sample whereinthe emitted fluorescent light is maximally optimized; in other words,little to none of the emitted light is reabsorbed by the fluorescentprotein. For this to work, the fluorescent protein and fluorophorefunction as an energy transfer pair wherein the fluorescent proteinemits at the wavelength that the acceptor fluorophore absorbs and thefluorophore then emits at a wavelength farther from the fluorescentproteins than could have been obtained with only the fluorescentprotein. Alternatively, the fluorophore functions as the energy donorand the fluorescent protein is the energy acceptor. Particularly usefulfluorescent proteins are the phycobiliproteins disclosed in U.S. Pat.Nos. 4,520,110; 4,859,582; 5,055,556 and the fluorophore bilin proteincombinations disclosed in U.S. Pat. No. 4,542,104. Alternatively, two ormore fluorophore dyes can function as an energy transfer pair whereinone fluorphore is a donor dye and the other is the acceptor dye(including any dye compounds disclosed in U.S. Pat. Nos. 6,358,684;5,863,727; 6,372,445 and 5,656,554).

Reactive Groups

Selected phosphate-binding compounds of the invention include one ormore reactive groups within their structure. The reactive group providesa locus for attaching a metal chelating moiety to another species,generally referred to herein as a “component” of a conjugate. Exemplarycomponents include biological or non-biological molecules, linkers,solid supports, phosphorylated target molecules and the like. Thereactive group reacts with a functional group of complementaryreactivity at the “attachment site” of the component of the conjugate.The reaction leads to the formation of a covalent linkage between themetal chelating moiety of the phosphate-binding compound and thecomponent of the conjugate.

Therefore, these reactive groups function to attach a biological ornon-biological component to the metal chelating moiety or are used toform a covalent attachment between the present chelating moieties andthe phosphorylated target molecules. Such components include solid andsemi-solid matrices such as polymeric particles (in particularpolystyrene microspheres of a diameter less that about 16 microns),magnetic particle, polymeric membranes and glass. These substances areparticularly useful when an assay is utilized wherein thephosphate-binding compound is immobilized, such as for isolationpurposes or when a further assay is conducted directly on an immobilizedphosphorylated target molecules, as described in this invention.

In addition, any biological component can be covalently attached by wayof reactive groups to the phosphate-binding compound; these include butare not limited to, proteins, peptides, saccharides and polysaccharides,nucleic acids (including nucleotides and nucleosides), amino acids,organelles, cells and cellular extract components.

Therefore, the phosphate-binding compounds of the present invention canalso comprise reactive groups, such as an amine-reactive group, for thecovalent attachment of the phosphate-binding compound to a matrix,microparticle, a phosphate target molecule or directly to a biologicalcomponent. Thus, when the ternary complex comprising thephosphate-binding compound, metal ion and phosphorylated targetmolecules forms, the reactive groups can form an additional covalentbond with the phosphorylated target molecule. This effectively increasesthe complex's stability and allows for more stringent isolation andanalysis of phosphorylated target molecules, including being able tomaintain the complex's integrity above the moderately acidic pH range.

Typically, covalent attachment of the phosphate-binding compound to amolecule is the result of a chemical reaction between an electrophilicgroup and a nucleophilic group. However, in a preferred embodiment, whena reactive group is used that is photoactivated, the covalent attachmentresults when the binding solution is illuminated. This is particularlyadvantageous to ensure that only phosphorylated target molecules form acovalent attachment to the binding compounds of the present invention.Photoactivatable reactive groups include, without limitation,benzophenones, aryl azides and diazirines. Exemplary phosphate-bindingcompounds comprising a photoactivatable group include Compounds 34, 36,39, 42 and 44.

Typically, the conjugation reaction between the reactive group and thecomponent to be conjugated results in one or more atoms of the reactivegroup to be incorporated into a new linkage attaching the compound orreagents of the invention to the biological or non-biological component.Selected components include, without limitation, these other moleculesinclude without limitation, labels, biological components (proteins,nucleic acid,), non-biological components including microparticles,plastic such as microplate wells, polymers such as PVDF, nitrocellulose,polysaccharides in particular agarose, dextrans and cellulose includingany compounds disclosed in U.S. Pat. No. 5,453,517.

Selected examples of nucleophile, electrophiles and resulting covalentlinkages are shown in Table 1. TABLE 1 Examples of some routes to usefulcovalent linkages with electrophile and nucleophile reactive groupsElectrophilic Group Nucleophilic Group Resulting Covalent Linkageactivated esters* amines/anilines carboxamides acrylamides thiolsthioethers acyl azides** amines/anilines carboxamides acyl halidesamines/anilines carboxamides acyl halides alcohols/phenols esters acylnitriles alcohols/phenols esters acyl nitriles amines/anilinescarboxamides aldehydes amines/anilines imines aldehydes or ketoneshydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkylhalides amines/anilines alkyl amines alkyl halides carboxylic acidsesters alkyl halides thiols thioethers alkyl halides alcohols/phenolsethers alkyl sulfonates thiols thioethers alkyl sulfonates carboxylicacids esters alkyl sulfonates alcohols/phenols ethers anhydridesalcohols/phenols esters anhydrides amines/anilines carboxamides arylhalides thiols thiophenols aryl halides amines aryl amines aziridinesthiols thioethers boronates glycols boronate esters carboxylic acidsamines/anilines carboxamides carboxylic acids alcohols esters carboxylicacids hydrazines hydrazides carbodiimides carboxylic acids N-acylureasor anhydrides diazoalkanes carboxylic acids esters epoxides thiolsthioethers haloacetamides thiols thioethers halotriazinesamines/anilines aminotriazines halotriazines alcohols/phenols triazinylethers imido esters amines/anilines amidines isocyanates amines/anilinesureas isocyanates alcohols/phenols urethanes isothiocyanatesamines/anilines thioureas maleimides thiols thioethers phosphoramiditesalcohols phosphite esters silyl halides alcohols silyl ethers sulfonateesters amines/anilines alkyl amines sulfonate esters thiols thioetherssulfonate esters carboxylic acids esters sulfonate esters alcoholsethers sulfonyl halides amines/anilines sulfonamides sulfonyl halidesphenols/alcohols sulfonate esters*Activated esters, as understood in the art, generally have the formula—COΩ, where Ω is a good leaving group (e.g. succinimidyloxy (—OC₄H₄O₂)sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H), -1-oxybenzotriazolyl (—OC₆H₄N₃);or an aryloxy group or aryloxy substituted one or more times# by electron withdrawing substituents such as nitro, fluoro, chloro,cyano, or trifluoromethyl, or combinations thereof, used to formactivated aryl esters; or a carboxylic acid activated by a carbodiimideto form an anhydride or mixed anhydride —OCOR^(a) or —OCNR^(a)NHR^(b),where R^(a) and R^(b), which may be the same or different, # are C₁-C₆alkyl, C₁-C₆ perfluoroalkyl, or C₁-C₆ alkoxy; or cyclohexyl,3-dimethylaminopropyl, or N-morpholinoethyl).**Acyl azides can also rearrange to isocyanates

Preferred reactive groups incorporated into the compounds of theinvention react with an amine, a thiol or an alcohol. In one embodiment,the reactive group is a photoactivatable group, an acrylamide, anactivated ester of a carboxylic acid, an acyl azide, an acyl nitrile, analdehyde, an alkyl halide, an amine, an anhydride, an aniline, an arylhalide, an aryl azide, an azide, an aziridine, a benzophenone, aboronate, a carboxylic acid, a diazoalkane, a diazirine, ahaloacetamide, a halotriazine, a hydrazine, an imido ester, anisocyanate, an isothiocyanate, a maleimide, a phosphoramidite, asulfonyl halide, or a thiol group.

Where the reactive group is an activated ester of a carboxylic acid, theresulting compound is particularly useful for preparing conjugates ofproteins, nucleic acids, e.g., nucleotides and oligonucleotides, orhaptens. Where the reactive group is a maleimide or haloacetamide theresulting compound is particularly useful for conjugation tothiol-containing substances. Where the reactive group is a hydrazide,the resulting compound is particularly useful for conjugation toperiodate-oxidized carbohydrates and glycoproteins, and in addition isan aldehyde-fixable polar tracer for cell microinjection. Where thereactive group is a silyl halide, the resulting compound is particularlyuseful for conjugation to silica surfaces, particularly where the silicasurface is incorporated into a fiber optic probe subsequently used forremote ion detection or quantitation or forms the substrate of amicroarray or biochip. Where the reactive group is a photoactivatablegroup (benzophenone, aryl azide or diazirine) the resultingphosphate-binding compound is particularly useful for conjugation tophosphorylated target molecules, See Example 50.

Preferably, the reactive group is a photoactivatable group, asuccinimidyl ester of a carboxylic acid, a haloacetamide, a hydrazine,an isothiocyanate, a maleimide group, an aliphatic amine, a silylhalide, or a psoralen. More preferably, the reactive group is aphotoactivatable group, a succinimidyl ester of a carboxylic acid, amaleimide, an iodoacetamide, or a silyl halide.

Linkers

In an exemplary embodiment, the compounds of the present invention thatinclude a reactive group or label further comprise a linker. The linkerserves to covalently attach the reactive group or label to the metalchelating moiety of the phosphate-binding compound. When present, thelinker is a single covalent bond or a branched- or straight-chain,saturated or unsaturated chain of atoms. Examples of L includesubstituted or unsubstituted polyalkylene, arylene, alkylarylene,arylenealkyl, or arylthio.

In certain embodiments, no linker is present when the phosphate-bindingcompounds comprise a label. In this instance the label and themetal-chelating moiety share an aromatic ring, e.g., benzofuran andBAPTA. Thus, when the label and chelating moiety share an aromatic ringno linker is present and n of the formula (A)m(L)n(B) is 0. A preferredembodiment, when the metal chelating moiety comprise a label, isphosphate-binding compounds wherein no linker is present; however,linkers as single covalent bonds are equally preferred.

Thus, the label or reactive group may be directly attached (where Linkeris a single bond) to the present compounds or attached through a seriesof stable bonds. When the linker is a series of stable covalent bondsthe linker typically incorporates 1-30, more preferably 1-20, and mostpreferred 1-15 non-hydrogen atoms selected from the group consisting ofC, N, O, S and P. In addition, the covalent linkage can incorporate aplatinum atom, such as described in U.S. Pat. No. 5,714,327.

The linker may be any combination of chemical bonds, optionallyincluding, single, double, triple or aromatic carbon-carbon bonds, aswell as carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygenbonds, sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygenbonds, phosphorus-nitrogen bonds, and nitrogen-platinum bonds. Exemplarycomponents of the linker include ether, thioether, thiourea, amine,ester, carboxamide, sulfonamide, hydrazide bonds and aromatic orheteroaromatic bonds.

An exemplary linker is a combination of single carbon-carbon bonds andcarboxamide, sulfonamide or thioether bonds. The bonds of the linkertypically result in the following moieties being in the linker: ether,thioether, carboxamide, thiourea, sulfonamide, urea, urethane,hydrazine, alkyl, aryl, heteroaryl, alkoxy, cycloalkyl and aminemoieties.

Any combination of linkers may be present in the presentphosphate-binding compounds. An exemplary compound of the presentinvention, when attached to more than one label, reactive group or acombination thereof will have one or two linkers attached that may bethe same or different. The linker may also be substituted to alter aphysical property of the present compounds, such as affinity,hydrophilicity, solubility and spectral properties of the compound.

Selected examples of phosphate-binding compounds incorporate thefollowing three (I, II and III) Linker formulas: Formula (I)—(CH₂)_(e)C(X)NH(CH₂)_(e)(NHC(X)(CH₂)_(e))_(d)— and Formula (II)—((C₆R″₄)O)_(d)(CH₂)_(e)(C(X)NH(CH₂)_(e))_(g)(NH)_(d)C(X)NH(C₆R″₄)(CH₂)_(e)—,Formula (III)—(NHC(X)(NH)_(d)(CH₂)_(e)(NH)_(d)C(X)(NH)_(d)(CH₂)_(e)(NHC(X)(CH₂)_(e))_(d))—,wherein X is O or S, d is 0-1, e is 0-6, g is 1-4 and R″ isindependently H, halogen, alkoxy or alkyl. It is understood that X, d, eand g are independently selected within a linker.

Thus, a selected embodiment of the present invention is the followingphosphate-binding compound formulas (VII, VIII, IX, X and XI): Formula(VII) (A)(B), no linker; Formula (VIII) (A)-(n)(B), linker is a singlecovalent bond; Formula (IX)(A)-[(CH₂)_(e)C(X)NH(CH₂)_(e)(NHC(X)(CH₂)_(e))_(d)]—(B); Formula (X)(A)-[(C₆R″₄)O)_(d)(CH₂)_(e)(C(X)NH(CH₂)_(e))_(g)(NH)_(d)C(X)NH(C₆R″₄)(CH₂)_(e)]—(B)and Formula (XI)(A)-[NHC(X)(NH)_(d)(CH₂)_(e)(NH)_(d)C(X)(NH)_(d)(CH₂)_(e)(NHC(X)(CH₂)_(e))_(d)]—(B),wherein A is a chemical moiety and B is a metal-chelating moiety.

Any combination of linkers may be used to attach the chemical moiety andthe metal-chelating moiety together. In addition, a metal-chelatingmoiety may have more than one linker that is used to attach eitheranother label, such as an energy transfer pair, or an additionalsubstance such as agarose, a microparticle or a reactive group thatfunctions to attach the linker to the additional substance or to thephosphorylated target molecule. A preferred embodiment includes ametal-chelating moiety attached to a label, with or without a linker,and also attached to a reactive group, a phosphorylated target molecule,a non-biological component or a biological component. The linker mayalso be substituted to alter the physical properties of thephosphate-binding compound, such as binding affinity of themetal-chelating moiety and spectral properties of the dye, orsubstituted with an amine- or thiol-reactive group.

Another important feature of the linker is to provide an adequate spacebetween the chemical moiety A and the chelating moiety B so as toprevent the chemical moiety from providing a steric hindrance to thebinding of the metal ion for the binding domain of the metal-chelatingmoiety and the binding of the metal ion for the phosphorylated targetmolecule. It is appreciated that not all chemical moieties will providea steric hindrance, as a preferred embodiment of the present inventionis a metal-chelating moiety that comprises a dye label without a linker.However, some labels such as biotin and reactive groups are typicallyattached to the metal-chelating moiety by a linker. Therefore, thelinkers of the present phosphate-binding compounds are important for (1)attaching the chemical moiety A to the metal-chelating moiety, (2)providing an adequate distance between the chemical moiety and themetal-chelating moiety so as not to sterically hinder the affinity ofthe metal-chelating moiety and a phosphate group on a target moleculeand (3) for altering the affinity of the metal-chelating moiety for thephosphorylated target molecule either by the choice of the atoms of thelinker or indirectly by addition of substituents to the linker.

The metal-chelating moieties of the present invention typicallycontain 1) no linker, 2) a single covalent bond as a linker, 3) a linkerof Formula I, 4) a linker of Formula II, 5) a linker of Formula III or acombination thereof. However, it is appreciated that a wide variety oflinkers that do not fall within the scope of these formulas are alsouseful as linkers of the phosphate-binding compounds. These options canbe present individually or in any combination, as embodied by theformula (A)m(L)n(B), on the metal-chelating moiety to attach chemicalmoieties A such as labels or reactive groups to form thephosphate-binding compounds of the present invention.

Synthesis

The synthetic strategy of phosphate-binding compounds that provideoptimal signals after formation of a ternary complex involves selectionof appropriate chemical linkages between the chemical moieties A and themetal-chelating moiety, and also selection of appropriate substituentson the metal-chelating moiety. These selections are made such that theresulting phosphate-binding compound retains optimal simultaneousbinding affinity for both the metal and of the metal for thephosphorylated target molecules and sufficient solubility to promote apersistent ternary complex. Improper selections result inphosphate-binding compounds that do not have sufficient binding affinityand do not produce a persistent ternary complex. Improper selectionsalso result in excessive non-selective binding of the phosphate-bindingcompound to analytes other than the phosphorylated target compounds,resulting in a high background and thus a low signal-to-noise ratio.Compounds that are suitable for practice of the invention are bestscreened by the method in Example 1D.

We have discovered that BAPTA derivatives are particularly suitable forpractice of the various aspects of the invention, although other metalchelating moieties under appropriate conditions are equally preferred.The novel phosphate-binding compounds of the present invention whosesynthesis and use is illustrated in examples include BAPTA chelatingmoieties with a quinazolinone fluorescent dye (Compounds 6, 7 and 23),BAPTA chelating moieties with a borapolyazaindacene fluorescent dye(Compounds 8, 24 and 27a-f), BAPTA chelating moieties with a xanthenebased dye (Compounds 11, 19, 26 and 25) BAPTA chelating moieties with abiotin label, wherein the biotin is attached by a linker (Compounds 9,12, 15 and 18), BAPTA chelating moieties with a benzothiazole label(Compound 17), BAPTA chelating moieties with agarose covalently attached(Compounds 13 and 14), BAPTA compounds comprising an aniline attached bya linker to the BAPTA compound (Compound 10) and BAPTA chelatingmoieties with a label and a photoactivatable reactive group (Compounds34, 36, 39, 42 and 44). Novel compounds also include borapolyazaindacenefluorophore labels attached by a linker to DTPA chelating moieties(Compounds 20, 21 and 22). These novel phosphate-binding compounds finduse in the detection and isolation of phosphorylated target molecules.Synthesis of these compounds is exemplified in Examples 30-48 and 52-56.

The phosphate-binding compounds of the present invention exhibitsufficient noncovalent binding affinity for the gallium(III)-phosphorylated target molecule complex to allow for rinsing awayof excess reagents from the persistent ternary complex. Additionally, itwas found that certain phosphate-binding compounds provided optimalsignal after formation of the ternary complex and are thus moreenvironmentally sensitive. This high signal appears to be a function ofwell-tuned hydrophobicity of the phosphate-binding compound—gallium(III)-phosphorylated target molecule complex. Therefore, when adetectable response is desirable, e.g., labeling phosphorylated targetmolecules in solution, and where the detectable response is afluorescence response, it is typically a change in fluorescence, such asa change in the intensity, excitation or emission wavelengthdistribution of fluorescence, fluorescence lifetime, fluorescencepolarization, or a combination thereof. Typically this change influorescence is a result of energy transfer between a first and secondfluorophore wherein an energy transfer pair is employed, one attached tothe chelating moiety and the other attached to a phosphorylatedmolecule, resulting in a shifted wavelength or quenching of thefluorescent signal. Preferably, when energy transfer is not employed,the detectable optical response upon binding the gallium ion and thephosphorylated target molecule to the chelator is a change influorescence intensity that is greater than approximately 2-fold.

However, for applications wherein the phosphorylated target molecule orphosphate-binding compound is immobilized—resulting in an immobilizedternary structure—an increase in detectable fluorescence response due tothe chelation of the metal-chelating moiety and subsequent ternarycomplex formation may not be necessary. This is due to the stableternary complex, which allows for washing and removal of unboundphosphate-binding compounds wherein the fluorescence response from thephosphate-binding compound is sufficient to visualize the phosphorylatedtarget molecule. Therefore, a preferred embodiment in this situation isa phosphate-binding compound that undergoes little or no change influorescence when bound to a metal ion of the present invention and aphosphorylated target molecule.

The combination of metal-chelating moieties, labels and or reactivegroups provides phosphate-binding compounds that are eitherenvironmentally sensitive (i.e., that produce a fluorescence change uponsimultaneously binding a metal ion and the phosphorylated targetmolecules to form a ternary complex) or insensitive (i.e., that producesno change in fluorescence signal). The most preferred fluorescent dyesof the present invention for generating a strong detectable signal andfacilitating formation of the ternary complex include benzofuran,quinoline, quinazolone, xanthene, benzazole, and borapolyazaindacenecompounds including various derivatives thereof. These fluorescent dyesproduce a strong detectable signal when the dye comprises ametal-chelating moiety. It is an important aspect of the currentinvention that none of the preferred fluorescent dyes are sulfonated.TABLE 2 Compound number Phosphate-binding compound Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

Compound 15

Compound 16

Compound 20

Compound 21

Compound 22

Compound 29

Compound number Properties Compound 1 The fluorophore is a fluorinatedxanthene (fluorescein) derivative and the metal-chelating moiety is aBAPTA compound (Formula IV) that is fluorinated at the R⁶ position. Thelinker is a single covalent bond. The counterion is K⁺. Compound 2 Thefluorophore is a xanthene (rhodamine) derivative and the metal-chelating moiety is a BAPTA compound (Formula IV) that is fluorinated atthe R⁶ position. The linker is a single covalent bond. The counterion isK⁺. Compound 3 The fluorophore is a xanthene (rhodamine) derivative andthe metal- chelating moiety is a BAPTA compound (Formula IV). The linkeris a single covalent bond. The counterion is K⁺. Compound 4 Thefluorophore is a benzofuran that shares an aromatic ring with themetal-chelating moiety (Formula IV) and comprises a substitutedheteroaryl moiety. The metal-chelating moiety, BAPTA, is fluorinated atthe R⁶ position. The phosphate-binding compound does not comprise alinker. The counterion is K⁺. Compound 5 The fluorophore is a xanthene(rhodamine) derivative and the metal- chelating moiety is a BAPTAcompound (Formula IV) that is fluorinated at the R⁶ position. The linkeris a single covalent bond. The counterion is K⁺. Compound 6 Thefluorophore is a quinazolinone with an adjacent hydroxyl group on themetal-chelating moiety (BAPTA, Formula IV). R¹³ and R¹⁴ areindependently hydrogen or a salt and the linker is a single covalentbond. Compound 7 The fluorophore is a quinazolinone with an adjacenthydroxyl group on the metal-chelating moiety (BAPTA, Formula IV). Themetal-chelating moiety is fluorinated at the R⁶ position and R¹³ and R¹⁴are independently hydrogen or a salt. The linker is a single covalentbond. Compound 8 The fluorophore is a borapolyazaindacene and the metal-chelating moiety is a BAPTA compound (Formula IV). The metal- chelatingmoiety is fluorinated at the R⁶ position and R¹³ and R¹⁴ areindependently hydrogen or a salt. The linker is represented by FormulaI. Compound 9 The fluorophore is a xanthene (rhodamine) derivative andthe metal chelating moiety is a BAPTA compound (Formula IV), wherein R¹³and R¹⁴ are independently hydrogen or a salt. The linker attaching thefluorophore to the BAPTA compound is a single covalent bond. A secondlinker at R⁷ (Formula I) covalently attaches a biotin label to thephosphate-binding compound. Compound 10 The fluorophore is a xanthene(rhodamine) derivative that is attached by a single covalent bond linkerto the metal-chelating moiety (BAPTA, Formula IV), wherein R¹³ and R¹⁴are second linker (Formula I) at R⁷ independently hydrogen or a salt. Aattaches an aniline moiety. Compound 11 The fluorophore is a xanthene(rhodamine) derivative that is attached to the metal-chelating moiety(BAPTA, Formula IV) by a single covalent bond, wherein R¹³ and R¹⁴ areindependently hydrogen or a salt. A second linker (Formula I) at R⁷attaches an amine group. Compound 12 The label is a biotin that isattached to the metal-chelating moiety (BAPTA, Formula IV) by a linker(Formula III). The metal-chelating moiety is fluorinated at the R³position and R¹³ and R¹⁴ are independently hydrogen or a salt. Compound13 The metal-chelating moiety (BAPTA, Formula IV) is attached to agaroseby a linker, wherein R¹³ and R¹⁴ are independently hydrogen or a salt.Compound 14 The metal-chelating moiety (BAPTA, Formula IV) is attachedto agarose by a linker, wherein R¹³ and R¹⁴ are independently hydrogenor a salt and the metal-chelating moiety is fluorinated at the R³position. Compound 15 The metal-chelating moiety (BAPTA, Formula IV) issimultaneously attached to biotin and a xanthene (rhodamine) derivativefluorophore, both by a linker represented by Formula III. R¹³ and R¹⁴are independently hydrogen or a salt. Compound 16 The metal-chelatingmoiety (BAPTA, formula IV) is attached by a single covalent bond to axanthene (rhodamine) derivative. R¹³ and R¹⁴ are independently hydrogenor a salt. Compound 20 The dye is a borapolyazaindacene that is attachedto the metal-chelating moiety (DTPA, Formula V) by a linker (FormulaII). The dye is substituted by a thienyl group. The counterion is K⁺.Compound 21 The dye is a borapolyazaindacene that is attached to ametal-chelating moiety (DTPA, Formula V) by a linker (Formula II). Thecounterion is K⁺. Compound 22 The dye is a borapolyazaindacene hat isattached to the metal-chelating moiety (DTPA, Formula V) by a linkerrepresented by Formula II. The counterion is K⁺. Compound 29 The dye isa xanthene derivative that is attached to the metal-chelating moiety(Formula IV) by a linker that is a single covalent bond. The counter-ion is K⁺.Binding Solution

The present binding solution comprising:

-   -   a) a metal chelating moiety;    -   b) a salt comprising trivalent metal ions, wherein said metal        ion is capable of simultaneously binding said metal chelating        moiety and a phosphorylated target molecule; and,    -   c) an acid.

The metal chelating moiety is optionally attached to a label, a reactivegroup or a combination thereof wherein the metal chelating moiety istypically selected from the group consisting of BAPTA, IDA, DTPA andphenanthrolines. However, any metal chelating moiety described above,specifically or generically, is considered part of the invention to beused in the present binding solution. The label is typically a memberselected from the group consisting of a dye, an enzyme and a hapten andthe reactive group is preferably a photoactivatable group. In apreferred embodiment the dye is selected from the group consisting of abenzofuran, a quinazolinone, a xanthene, an indole, a benzazole and aborapolyazaindacene provided that said dyes are not sulfonated.

Thus, in a preferred embodiment, the binding solution of the presentinvention comprises the following components:

-   -   a) a phosphate-binding compound having formula (A)m(L)n(B)        wherein A is a chemical moiety, L is a linker, B is a        metal-chelating moiety, m is an integer from 1 to 4 and n is an        integer from 0 to 4;    -   b) a salt comprising metal ions; and,    -   c) an acid.

The binding solution can be prepared in a variety of ways, which aredependent on the method and the medium in which the sample is present,as described below. In a preferred embodiment, the binding solutioncomprises a phosphate-binding compound having formula (A)m(L)n(B), asalt comprising a metal ion and acid in an aqueous solution sufficientto adjust the pH of the binding solution to 3-6; optionally the bindingsolution comprises an organic solvent or a mixture of organic solventsand additional ionic or nonionic components, e.g. sodium chloride. Anyof the components of the binding solution can be added together orseparately and in no particular order and, as will become evident, thephosphate-binding compound may be immobilized on a solid or semi-solidmatrix, wherein the metal ion and acid are added to the matrix to formthe binding solution of the present invention. Therefore, thephosphate-binding compounds do not need to be free in the bindingsolution to form the solution but may be immobilized on a solid orsemi-solid matrix surface. We have found that depending on the method,i.e. detection and isolation, that the concentration of metal ion andphosphate-binding compound needs to be adjusted.

Soluble phosphate-binding compounds are prepared by dissolution in asolvent, such as water, DMSO, DMF or methanol, usually at a finalconcentration of about 0.1 μM to 10 μM; preferably, thephosphate-binding compound is present in the binding solution at aconcentration of about 0.5 μM to 5 μM and most preferably at aconcentration of about 1.0 μM. However, in applications in which thebinding solution is used to precipitate phosphorylated target moleculesfrom solution, a higher concentration of phosphate-binding compounds inthe binding solution is desired—preferably about 0.05 mM to 1 mM. Forprecipitation purposes that concentration is increased but the ratio ofphosphate-binding compound to metal ion is comparable to the ratio ofthe binding solution used for detection purposes.

The metal ion-containing salt preferably contains trivalent galliumions, such as is prepared from gallium chloride, but can be any galliumsalt known to those skilled in the art. Alternatively iron and aluminumions also find use in the binding solution of the present invention.Gallium salts that can be used with the present invention include,without limit, acetylacetonate, arsenide, bromide, chloride, fluoride,iodide, nitrate, nitride, perchlorate, sulfate and sulfide. The galliumsalt is typically present in the binding solution at a concentration ofabout 10 nM to about 1 mM; preferably the concentration of the galliumsalt is about 0.5 μM to 10 μM. However, for precipitation purposes, thegallium salt is preferably present at a slightly higher concentration ofabout 0.1 mM to about 0.5 mM.

Analysis of the stability and specificity of the phosphate-bindingcompounds for gallium ions and the gallium ions for the phosphorylatedtarget molecules was evaluated as a function of pH (Example 1). Based onthese results, it was determined that a preferred binding solutioncomprises an acid to provide a moderately acidic environment for thebinding reaction. In fact, an important and unexpected aspect of thepresent invention is that metal-chelating groups bind trivalent cationssuch as gallium in a moderately acidic environment, resulting in atitration of fluorescent signal with an increase in pH level approachingneutral pH. An acidic environment is defined as a solution having a pHless than 6.9. Typical suitable acidic components include withoutlimitation acetic acid, trichloroacetic acid, trifluoroacetic acid,perchloric acid, or sulfuric acid. The acidic component is typicallypresent at a concentration of 1%-20% and is buffered to the appropriatepH by a base. The pH of the binding solution is preferably about pH 3-6and most preferred is about pH 4.0. Acetic acid is a preferred acid foruse at or near pH 4. The optimal pH for each compound used may varyslightly depending on the compound used; for Compound 2, pH 4.0 ispreferred.

The pH of the binding solution is optionally modified by the inclusionof a buffering agent in addition to the acidic component. In particular,we have shown that the presence of a buffering agent unexpectedlyimproves binding of phosphorylated target molecules immobilized inelectrophoresis gels, provided that an alcohol is also included in theformulations. Any buffering agent that maintains an acidic environmentand is compatible with the phosphorylated target molecules in the sampleis suitable for inclusion in the binding solution.

Useful buffering agents include salts of formate, acetate,2-(N-morpholino)ethanesulfonic acid, imidazole,N-(2-hydroxyethyl)piperazinylethanesulfonic acid,tris-(hydroxymethyl)aminomethane acetate, ortris(hydroxymethyl)aminomethane, hydrochloride, wherein the bufferingagent does not chelate gallium ions. An exemplified buffering agent issodium acetate. The buffering agent is typically present in the bindingsolution at a concentration of about 20 mM to 500 mM; preferably theconcentration is about 50 mM to 200 mM.

Inclusion of a water-miscible organic solvent, typically an alcohol, inthe binding solution is recommended when the binding solution contains apH-buffering agent and a salt. Although the use of highly polar solventssuch as formamide is permitted, typically, the polar organic solvent isan alcohol having 1-6 carbon atoms, or a diol or triol having 2-6 carbonatoms. A preferred alcohol is 1,2-propanediol. The polar organicsolvent, when present, is typically included in the binding solution ata concentration of 5-50%. The presence of a polar organic solvent isparticularly advantageous when binding sodium dodecyl sulfate(SDS)-coated proteins, as is typically the case when bindingphosphorylated proteins or peptides that have been electroblotted fromSDS-polyacrylamide gels. Typically, in the preferred procedure, SDS isremoved from a gel or blot prior to addition of the binding solution byfixing and washing; however, some SDS may remain and can interfere withthe binding methods of the present invention. Without wishing to bebound by any theory, it appears that the presence of an alcohol improvesluminescent labeling of phosphorylated proteins or peptides by removingany SDS that was not removed by washing or fixing the sample. However,nitrocellulose membranes may be damaged by high concentrations ofalcohol (for example, greater than about 20%), and so care should betaken to select solvent concentrations that do not damage the membranesupon which the phosphorylated proteins or peptides are immobilized.

Methods of Use

The phosphate-binding compounds of the present invention can be usedwithout limitation for the analysis and monitoring of phosphorylatedtarget molecules. In this way, phosphorylated target molecules can bedetected in unlimited assay formats that provide information about thenumber of phosphate groups on the target molecule, the identification ofenzymes involved in phosphorylation and dephosphorylation, the role thatsuch target molecules have in the proteome and—with further analysis—thesite of attachment of phosphate groups on the target molecules. Furtheranalysis can be carried out after the compounds of the present inventionare used to selectively detect and/or isolate phosphorylated targetmolecules.

The methods of the present invention can be carried out on samples thatare immobilized, on samples in which the phosphate-binding compound isimmobilized or where both the sample and phosphate-binding compounds arein solution. The binding solution is combined with the sample in such away as to facilitate contact between the phosphate-binding compound,trivalent metal ion and any phosphorylated target molecules present inthe sample, wherein formation of a ternary complex effectively binds achemical moiety A to the phosphorylated target molecules that arepresent. When the sample is immobilized on a solid or semi-solidsupport, the binding solution is typically incubated with the sampleunder conditions that maximize contact, such as gentle mixing orrocking.

The methods of the present invention for detecting phosphorylated targetmolecules that have been immobilized on a gel comprise the followingsteps:

-   -   i) immobilizing the sample on a gel;    -   ii) optionally contacting the gel of step i) with a fixing        solution;    -   iii) contacting the gel of step ii) with a binding solution of        the present invention    -   iv) incubating the gel of step iii) and the binding solution for        sufficient time to allow said compound to associate with said        phosphorylated target molecule;    -   v) visualizing the phosphate-binding compound whereby said        phosphorylated target molecule is detected; and,    -   vi) optionally, a second (or third) stain is added to the gel to        detect either total protein or proteins of another class, such        as glycoproteins, or both.

Typically, immobilizing the sample on a gel compriseselectrophoretically separating the sample. The gel, without limit,includes any gel known to one of skill in the art for separating targetmolecules from each other, including polymer-based gels such as agaroseand polyacrylamide wherein an electrical current is passed through thegel and the target molecules migrate based on charge and size. Thus,gels (reduced and native) also include both one and two-dimensionalgels, and isoelectric focusing gels. Capillary electrophoresis may beemployed using gels, solutions containing polymers, or solutions alone.

Optionally, a sample separated on a gel may be transferred to apolymeric membrane, using techniques well known to one skilled in theart, wherein the membrane is then contacted with a binding solution ofthe present invention to selectively detect phosphorylated targetmolecules. A method of the present invention for detectingphosphorylated target molecules immobilized on a membrane comprises thefollowing steps:

-   -   i) electrophoretically separating the sample on a gel;    -   ii) transferring the separated sample to a membrane;    -   iii) optionally contacting the membrane of step ii) with a        fixing solution;    -   iv) contacting the membrane of step iii) with a binding        solution;    -   v) incubating the membrane of step iv) and the binding solution        for sufficient time to allow the compound to associate with the        phosphorylated target molecule; and,    -   v) visualizing the compound, whereby said phosphorylated target        molecule is detected.    -   vi) Optionally, a second (and/or third) stain is added to the        membrane to detect either total protein or proteins of another        class, such as glycoproteins.

Protein gel electrophoresis is typically performed using SDS as acomponent of either the sample preparation or in the running buffer.However, SDS interferes with the binding solution of the presentinvention and therefore must be removed from the gel or membrane priorto addition of the binding solution. Gels and membranes are fixed andwashed, which results in the removal of most or all of the SDS from thegels or blots. A preferred fixing solution for gels and membranescomprises methanol and acetic acid; optionally the fixing solutioncomprises glutaraldehyde. The methanol is present at a concentration ofabout 35-50% and the acetic acid is present at about 0-15% and theglutaraldehyde is present at about 0-2%. Typically, washing the gels ormembranes with 100% water follows fixing.

However, for purposes of the invention, the binding solution alsodetects phosphorylated target molecules that have been separated on anative or non-reduced gel. Therefore, for methods utilizing these gelsthat do not contain SDS, the fixing solution step is not necessary.

After samples have been separated on a gel or transferred to a polymericmembrane, optionally fixed, and washed, the gel or blot is incubatedwith a binding solution (Examples 2-9). The phosphorylated proteins orpeptides are incubated with the binding solution for a time sufficientfor the phosphate-binding compound/metal ion complex to bind to thephosphorylated proteins or peptides that are present. Preferably, thistime is not more than 24 hours, more preferably this time is less than 8hours and most preferably this incubation time is less than 2 hours, butnot less than 5 minutes. After incubation with the binding solution thegels or membranes are typically washed with a mixture that preferablycomprise an acidic buffering agent and acetonitrile; useful bufferingagents to be used with the present invention include, withoutlimitation, NaOAc, formate and 2-(N-morpholino)ethanesulfonic acid.Typically, the buffering agent is present in the washing solution at aconcentration of about 25 mM to about 100 mM. In addition, it has beenfound that optional inclusion of acetonitrile in the washing solutionusually reduces non-specific labeling. Preferably, acetonitrile ispresent at a concentration from 1-7%, more preferably 3-4%. Analternative washing solution is comprised of 10-20% 1,2-propanediol.

Thus, following binding of the phosphate-binding compound and washing,the ternary complex can be illuminated directly when thephosphate-binding complex comprises a fluorophore or chromophore label,as described above, to visualize the phosphorylated target molecules.Alternatively the presence and location of the phosphorylated targetmolecule on the blot can be detected using antibodies to the label, suchas anti-BAPTA antibody, an anti-fluorophore antibody, an anti-haptenantibody or an avidin (when the label is a biotin derivative), which isthen detected by standard means used to detect proteins on Western blotssuch as by fluorescence, chemiluminescence or radioactivity, indicatinglabeling of the phosphorylated target molecules.

The phosphate-binding compounds of the binding solution are chosendepending upon their ability to bind phosphorylated target molecules indifferent media. Therefore, preferred phosphate-binding compounds forbinding phosphorylated target molecules in a gel include compounds 1-4and 7-11 of the present invention. Preferred compounds for bindingphosphorylated target molecules on a membrane include compounds 1, 4 and7 of the present invention.

A particular advantage to identifying phosphorylated proteins orpeptides in a 2-D gel is the ability to correctly identify thephosphoproteome, as well as to quantitate post-translationalmodification of proteins for the addition or subtraction of phosphategroups. Specifically, labeling of phosphorylated proteins or peptideswhile doing concurrent, or subsequent, total protein staining identifiesthe phosphorylated proteome, while the intensity of the signal can becorrelated to the level of phosphorylation, when compared to the totalprotein stain (see, Examples 6, 7 and 13). Any fluorescent dye specificfor total proteins can be used to stain total proteins in the gel; apreferred stain is SYPRO® Ruby dye for gels or any dye disclosed in U.S.Pat. No. 6,316,276 B1. Other fluorescent dyes such as MDPF and CBQCAcould also be used for detection on membranes. Because SDS is removed bywashing prior to staining with the staining mixture of the presentinvention, total protein stains such as SYPRO® Ruby dye are preferredbecause SDS is not critical for their staining function. However,protocol changes can be made when using a stain that requires SDS forstaining sensitivity, such as SYPRO® Orange dye, SYPRO® Red dye andSYPRO® Tangerine dye, by adding SDS back to the gel prior to a totalprotein stain step and including SDS in the staining solution for thetotal protein stain (Malone et al. Electrophoresis (2001) 22(5):919-32).A preferred mixture for returning SDS back to a gel is 2% acid/0.0005%SDS, and optionally 40% ethanol, wherein the gel is incubated for atleast one hour. Alternatively, the total protein stain can be performedprior to the phosphorylated target molecules staining of the presentinvention; therefore, in this case, it is not necessary to add back theSDS to the gel, but simply to remove the SDS prior to the phosphorylatedtarget molecule staining step, as contemplated by the present invention.Therefore, alternative preferable total protein stains for gels includebut are not limited to, SYPRO® Orange dye, SYPRO® Tangerine dye andSYPRO® Red dye or any dye disclosed in U.S. Pat. No. 5,616,502 or U.S.Ser. No. 09/632,927. Alternative, but less preferred, total proteinstains for gels include Coomassie Blue or silver staining, which utilizestaining techniques well known to those skilled in the art. Alternativetotal proteins stains useful for staining blots are SYPRO® Rose Plus dyeand DyeChrome™ dye or any dye solution disclosed in U.S. Pat. No.6,329,205 B1 and U.S. Ser. No. 10/005,050.

Another very important advantage when labeling phosphorylated targetmolecules in a 2-D gel is to include a stain for glycoproteins, whereina 3-way analysis of the proteome could be accomplished (Steinberg etal., “Rapid and Simple Single Nanogram Detection of Glycoproteins inPolyacrylamide Gels and on Electroblots,” Proteomics 1:841-855 (2001)).A preferred glycoprotein stain is Pro-Q™ Emerald 300 dye or Pro-Q™Emerald 488 dye, Pro-Q™ Fuchsia dye or any other dye disclosed in U.S.Ser. No. 09/970,215. In addition, if the sample comprises fusionproteins with oligohistidine affinity peptides, Pro-Q™ Sapphire 365 or488 dye can be used to simultaneously detect these proteins or peptides.

Thus, it is particularly advantageous that the parallel determination ofboth protein expression levels and functional attributes of the proteinssuch as phosphorylation of proteins can be achieved with the presentinvention within a single 2-D gel electrophoresis experiment. Analysiscan be accomplished by using image analysis software, e.g., Compugen'sZ3 program or Phoretix Progenesis software. Any two images can bere-displayed, allowing visual inspection of the differences between theimages, and quantitative information can be readily retrieved in tabularform with differential expression data calculated.

Alternatively, single-dimension polyacrylamide and corresponding blotscan be simultaneously or subsequently stained for total proteins orglycoproteins using staining techniques and dyes described above. Aparticular advantage for counterstaining a gel or blot that has beenlabeled using methods of the present invention is the ability todistinguish between nonspecific labeling and labeling of phosphorylatedtarget molecules with a low number of phosphate groups. This isimportant for accurately identifying phosphorylated target moleculesthat have undergone a small change in the degree of phosphorylation.Counterstaining a blot or gel with a total protein stain such as SYPRO®Ruby permits a ratiometric analysis of the fluorescent signal generatedfrom the dyes of the present invention compared to the fluorescentsignal generated from a total protein stain (see, FIG. 11 and Example22). This ratiometric analysis also permits the stoichiometrydetermination of the phosphorylated target molecule relating to theoverall phosphorylation state of the molecule as well as the addition orsubtraction of phosphate groups.

Another particular advantage for staining phosphorylated proteins orpeptides separated in polyacrylamide gels is for the analysis ofproteins of interest by combining spot detection with the compounds ofthis invention with mass spectrometry techniques for further analysis.For example, because phosphoproteins may co-migrate in a gel, furtheranalysis may be essential or desired to specifically identify andanalyze the phosphoprotein of interest. This further analysis can beachieved by measurement of a set of peptide masses derived from aprotein, i.e., by peptide mapping with mass spectrometry (MS), or byobtaining amino acid sequence information from individual peptides,i.e., protein sequencing by MS/MS or by Edman degradation. Thus, aprotein band or spot, once identified using the compositions and methodsof the present invention, may be excised from the gel, rinsed,optionally reduced and S-alkylated, and then digested in situ in the gelwith a sequence-specific protease, e.g., trypsin, using standardprotocols. See Shevchenko et al., “Mass Spectrometric Sequencing ofProteins from Silver Stained Polyacrylamide Gels,” Anal. Chem. 68:850-58(1996). The peptide mixture thus generated may be extracted from the geland analyzed by MS, using standard protocols. Peptide mapping bymatrix-assisted laser desorption/ionization (MALDI) mass spectrometry isoften most sensitive. Methods for the in-gel digestion of proteins aredescribed in Jensen et al., “Mass Spectrometric Identification andMicrocharacterization of Proteins From Electrophoretic Gels: Strategiesand Applications,” PROTEINS: Structure, Function, and Genetics Suppl.2:74-89 (1998).

DNA-binding proteins are key to the regulation and control of geneexpression, replication and recombination. The electrophoretic mobilityshift assay (or gel shift assay) is considered an essential tool inmodern molecular biology for the study of protein-nucleic acidinteractions. Nucleic acids could be detected with SYBR® Green II dye,while phosphoproteins are subsequently detected by methods described inthis invention. All fluorescence staining steps would be performed afterthe entire gel-shift experiment is completed, so there is no need topre-label either the DNA or the protein and no possibility of thefluorescent reagents interfering with the protein-nucleic acidinteractions. A third total protein stain might be employed as well,such as SYPRO® Ruby dye. In this way the influence of proteinphosphorylation on DNA-binding may be measured. The ability toindependently quantify each molecular species allows more rigorous dataanalysis methods to be applied, especially with respect to the mass ofphosphoprotein bound per nucleic acid.

The present invention is also contemplated to be used in a wide range ofmicroarray formats, including but not limited to the methods and arraysdisclosed in US Patent Application 2002/0076727; US Patent Application2002/0106785; US Patent Application 2002/0055186; WO 99/39210; WO00/63701; WO 02/25288; WO 01/18545, WO 00/04380 and U.S. Pat. Nos.6,403,368; 6,475,809; 6,365,418; 6,409,921; 5,595,915; 6,461,807;6,399,299. Phosphorylated target molecules immobilized on an array suchas a HydroGel-coated slide including those disclosed in U.S. Pat. Nos.6,372,813; 6,391,937; 6,387,631; 6,413,722 and those manufactured byPerkin Elmer; can also be detected using the methods and compositions ofthe present invention (Examples 18 and 19). Alternatively,phosphate-binding compounds can be immobilized on these arrays.

The methods of the present invention for detecting phosphorylated targetmolecules on an array typically comprise the steps of:

-   -   i) immobilizing said sample on an array;    -   ii) contacting said array of step i) with a binding solution,    -   iii) incubating said array of step ii) and said binding solution        for sufficient time to allow said compound to associate        indirectly with said phosphorylated target molecule; and,    -   iv) illuminating said compound with a suitable light source        whereby said phosphorylated target molecule is detected.

The sample is immobilized on the array using techniques well known toone skilled in the art, including but not limited to, using a piezoarray printer, contact printer or other array printer technology,immobilizing a phosphorylated target molecule, binding molecule such asan antibody and then added the sample to non-covalently bind thephosphorylated target molecules to the array. Typically the arraycomprises molecules that covalently attach the sample, or a protein thatselectively binds the sample, such as an amine-reactive group.

The array is incubated with a binding solution for sufficient time toform a ternary complex between a phosphate-binding compound, the metalion (typically gallium) and phosphorylated target molecule.Alternatively, the array may comprise phosphate-binding compoundscomplexed with the metal ions immobilized on the surface of the array,wherein a sample is incubated with the array and detection ofphosphorylated target molecules occurs when the target molecules bindthe metal ion/phosphate-binding complex and are typically illuminated,unbound sample is washed away. In this way, an assay to detectphosphatases or kinases is performed with an appropriate peptide orprotein substrate and the resulting phosphorylated or dephosphorylatedpeptides or proteins are spotted or synthesized on the array, whereinphosphate groups on the peptides bind the phosphate-bindingcompounds/metal-ion complex on the array. Alternatively, a kinase and/orphosphatase substrate is spotted or synthesized on the array and thenthe enzyme, kinase (and ATP) or phosphatase, is added to the array.After removing the enzyme, the array is then contacted with the bindingsolution. In this way, the array is used to detect and/or isolatephosphorylated target molecules and to identify the enzymes responsiblefor adding and/or removing phosphate groups from target molecules andtheir efficiency in doing so.

Typically phosphatase and kinase peptide substrates are immobilized onan array by spotting or synthesis using standard protocols, thephosphates or kinase enzymes, either comprise an unknown sample or areisolated enzymes, are added and subsequent presence of phosphate groupsis detected using a binding solution of the present invention. Thus, themethods and binding solution of the present invention are useful, forexample, with arrays of protein substrates for various protein kinases(e.g., myosin light chain, MARCKS, myelin basic protein, casein,src-supressed C kinase substrate, insulin Receptor Substrate 1, Nuclearfactor 90, Rap1, transcription factor stat5a). A sample comprisingphosphatase or kinase enzymes is incubated with the array comprisingenzyme substrate; following incubation under appropriate conditions andwith appropriate reaction additives for the enzymes the phosphorylatedproducts can be detected with a binding solution of the presentinvention. When the detectable label is a fluorophore, for example, thecoordinates of the fluorescent signals provides a read-out of thekinases present in the fluid and their activity against the variousenzyme substrates (peptides or proteins) on the array. Detection ofphosphorylated target molecules with an array offers many possibilitiesand the above description is not meant to limit how the presentinvention can be used in combination with array technology.

Current commercial kinase assays are often time-consuming and requiremany steps such as electrophoresis, centrifugation, ELISA orimmunoprecipitation. The present invention provides methods for therapid, sensitive, and non-radioactive detection of a variety of selectedkinases and phosphatases and provides, in addition, methods that arewell suited for high-throughput screening. The kinase and phosphataseassays of the present invention also permit the screening of inhibitorsand activators of, for example, tyrosine kinases and, in addition, alsopermit the monitoring and the purification of kinase and phosphataseenzymes. Moreover, detection of the enzyme substrate on the array makesthe methods of the invention far more sensitive than any knownsolution-based assays for kinases and phosphatases and use offluorescence or chemiluminescence for detection on the array permits ahigher density of labeling than is possible with radiochemicaldetection.

As described above, a kinase substrate is covalently or non-covalentlyattached to a surface, solid or semisolid matrix including a microwellplate, polymeric beads or an array such as a HydroGel array slide (oramine microarray substrate, aldehyde microarray substrate, an epoxymicroarray substrate, a poly-L-lysine microarray substrate or otherpolyacrylamide microarray substrates) and the assay is performed in anon-continuous heterogeneous manner. The kinase substrate comprises akinase consensus phosphorylation site, preferably a peptide or a randompolymer (poly(Glu:Tyr), poly(Glu:Ala:Tyr). Optionally the kinasesubstrate comprises a fluorophore. A sample suspected of containing akinase is combined with the kinase substrate, along with ATP, wherein anactive kinase enzyme will add phosphates to the kinase substrate. Theaddition of phosphate groups is measured after removal of the kinasesolution and adequate washing, wherein a binding solution, as describedabove, is added to the kinase substrate. Typically the phosphate-bindingcompound comprises a fluorophore and the kinase activity is measured byilluminating the fluorophore. Alternatively, the phosphate-bindingcompound comprises an enzyme such as peroxidase, wherein the kinaseactivity would be measured after addition of the appropriate enzymesubstrate and detection with a fluorometers or an instrument to measurecolor or chemiluminescence. In addition, using an inhibitor of theselected kinase or phosphatase in the assay, for example, by usingsodium orthovanadate may enhance the specificity of the kinase.Furthermore, the assay methods of this invention can be used to screenfor inhibitors or activators of kinases and/or phosphatases.Alternatively, the assay is easily adaptable to measure phosphataseactivity wherein the phosphatase substrate, phosphorylated peptides orproteins, would be bound to a solid or semi-solid matrix such as amicrowell plate, polymeric particle or a hydrogel.

The materials and methods of the present invention may also be used todetect and/or quantitate kinases or phosphatases by employing aFRET-based assay. For example, a peptide labeled with a fluorophore canbe combined with the phosphate-binding compound/metal-ion (typicallygallium) complex derivatized with a phycoerythrin or other dye label.When the peptide is phosphorylated, the peptide binds the dye labeledphosphate-binding compound and the emission maximum shifts in the assay.Time-resolved fluorescence can be achieved, for example, by employing aeuropium-based chelate on the peptide and the phosphate-bindingcompound/metal ion complex derivatized with allophycocyanin. The donorfluorophore can be excited, in this example, at 335 nm and an emissionshift from 620 nm to 665 nm indicates peptide interaction with themetal-chelator and gallium complex.

Thus, in one aspect of the invention, numerous enzymes, includingnitrogenase, phosphoribosyl-pyrophosphate synthetase, undecaprenylpyrophosphate synthase, DNA polymerases, RNA polymerases,farnesyltransferase, nucleoside triphosphate pyrophosphohydrolases,pyrophosphate-fructose 6-phosphate 1-phosphotransferase (PFPPT), sulfateadenyltransferase, UTP-glucose 1-phosphate uridinyltransferase (UGPP),asparagine synthetase, and UDP-glucose pyrophosphorylase involve themetabolism of inorganic pyrophosphate and thus are potential targets forquantitation by the disclosed invention.

In addition, the methods and materials of the present invention are alsouseful for studying functional proteomics involving ligand overlaymethodology. For example, arrayed proteins would be detected afterincubation with phosphatidylinositol 4,5-bisphosphate (PIP2) micelles,followed by incubation with the binding solution. The differences inlabeling would highlight an important class ofphosphatidylinositide-binding proteins. Proteins such as SWI/SNF-likeBAF, a chromatin remodeling complex and cofilin/ADF, a ubiquitousactin-binding protein, are likely to be identified using the methods ofthe present invention (Rando et al. Proc Natl Acad Sci USA 99(5):2824-9(2002); Ojala et al. Biochemistry 40(51):15562-9 (2001)). Anotherexample of a ligand overlay assay would be GTP-binding proteins, whereinthe small GTP-binding proteins can be separated by high-resolution 2-Dgel electrophoresis and subsequently transferred under renaturingconditions to a nitrocellulose or PVDF membrane and probed with GTP. Thebound GTP would then be subsequently bound with the binding solution ofthe present invention, resulting in identification of GTP-bindingproteins. A variety of other membrane overlay nucleotide-binding assayscould be preformed using the binding solution of the present invention,wherein potentially any ligand and binding protein, wherein at least oneof the pair contains phosphate group(s), could be used to identify novelbinding proteins (Gromov et al. Electrophoresis (1994) 3-4:478-81).

In contrast to having either the phosphorylated target molecules or thephosphate-binding compound immobilized, the compositions and methods ofthe present invention are also useful for binding, detecting andisolation of phosphorylated target molecules that are free in asolution. A sample suspected of containing phosphorylated targetmolecules is incubated with the binding solution comprising fluorescentdye-labeled phosphate-binding compounds wherein phosphorylated targetmolecules are detected by fluorescence polarization (Example 14) orenergy transfer (Example 51).

Fluorescence polarization is based upon the finding that the emission oflight by a fluorophore may be depolarized by rotational diffusion, orthe rate at which a molecule tumbles in solution (J. Phys. Rad.1:390-401 (1926)). Polarization is the measurement of the averageangular displacement of the fluorophore, which occurs between theabsorption and subsequent emission of a photon. This angulardisplacement of the fluorophore is, in turn, dependent upon the rate andextent of rotational diffusion during the lifetime of the excited state,which is influenced by the viscosity of the solution and the size andshape of the diffusing fluorescent species. If viscosity and temperatureare held constant, for example, then fluorescence polarization isdirectly related to the molecular volume or size of the fluorophore.Thus, when detecting phosphorylated target molecules in solution, thecompounds and methods of the present invention contemplate takingadvantage of fluorescent polarization, as described in U.S. Pat. No.6,207,397. The detection of phosphorylated target molecules would bebased upon the observation that changes in polarization occur when afluorescent dye-labeled phosphate-binding compound undergoes a molecularweight change due to the binding of a phosphorylated target molecule,for example, a phosphoprotein. The solution containing the sample andbinding solution are irradiated with plane-polarized light of awavelength that is sufficient to excite the fluorophore. The lightsubsequently emitted by the fluorescent phosphorylated target moleculeis polarized to varying degrees, depending on the molecular size of thefluorescent dye. In the unbound state in solution, low molecular weightlabeled phosphate-binding compounds will rotate rapidly, and give lowpolarization readings. The degree of polarization of the emission can bemeasured without the necessity to separate the components in thesolution. See, FIG. 10.

As discussed above for the kinase assay, FRET or energy transfer betweena first dye label and a second dye label can be employed for thedetection of phosphorylated target molecules in solution. A first dyelabel is employed on the phosphate-binding compound and a second dyelabel is added to the sample as part of a phosphorylated orphosphorylatable molecule. When the phosphate-binding compound binds thedye-labeled molecule then the first and second dye label are broughtwithin proximity that facilitates energy transfer. The first dye labelhas a first absorption and emission spectra and the second dye label hasa second absorption and emission spectra. Energy transfer occurs betweenthe two dye labels when overlap between the emission of the first dyelabel and the absorption of the second dye label is present wherein theabsorbed energy by the second dye label is either quenched or re-emittedat a longer wavelength. It is understood that the first dye label ispresent either as part of the phosphate-binding compound or as part of aphosphorylated or phosphorylatable target molecule; the same is also thecase for the second dye label.

In this instance, a method for detecting phosphorylated target moleculesin a solution sample comprises:

-   -   a) contacting said sample with a binding solution to form a        combined mixture, wherein said binding solution comprises a        phosphate-binding compound, a salt comprising trivalent metal        ions and an acid, wherein said combined mixture comprises a        first dye label that has a first absorption and emission spectra        and a second dye label that has a second absorption and emission        spectra;    -   b) incubating said phosphate binding compound and said sample        for a sufficient amount of time for said phosphate binding        compound to bind a phosphorylated target molecule; and,    -   c) illuminating said sample with an appropriate wavelength        whereby said phosphorylated target molecule is detected by a        change in fluorescence signal.

This method is particularly useful when the sample contains ATP, as isthe case for many kinase assays, due to the ability to discriminatebetween bound ATP and the kinase substrate. This method is also usefulwhen a phosphate-binding compound is employed that does not result in anincrease in signal intensity when a phosphorylated target molecule isbound compared to when no target molecule is bound. Thus, the use ofenergy transfer allows for a homogenous assay system that does notrequire separation or additional detection reagents for the detection ofphosphorylated target molecules in solution. For the detection ofimmobilized target molecules this is not an issue because unboundphosphate-binding compounds or unused ATP is washed away, leaving onlythe bound phosphate-binding compounds and a resulting detectable signal.However, we have found that for solution-based assays both fluorescencepolarization and FRET are preferred for the detection of phosphorylatedtarget molecules.

Thus in an aspect of the invention, the detection of kinase enzymes in asolution based assay comprises a dye-labeled kinase substrate, enzymeand ATP combined with a present binding solution wherein thephosphate-binding compound comprises a metal chelating moiety that iscovalently bonded to a dye label. The phosphate-binding compound maystill bind the free ATP but there will be no shift in signal, in thisinstance, a shift only occurs when a phosphate-binding compound binds aphosphorylated dye-labeled kinase substrate. This same methodology canbe applied to phosphatase substrates wherein the removal of phosphategroups from the substrate results in a signal from the first and seconddye label as would be observed without energy transfer. It isadvantageous that this assay be observed at several set time intervalsto observe the change in fluorescent signal and for a baseline signal.See, FIG. 12.

In a further aspect of the invention, the phosphate-binding compoundsthat comprise a reactive group can be used to covalently attach apresent phosphate-binding compound to a phosphorylated target molecule.The phosphate-binding compounds may further comprise a present label. Inthis instance, a binding solution is incubated with a phosphorylatedtarget molecule for a sufficient amount of time for thephosphate-binding compound to bind the target molecule. The covalentbond forms when an appropriate reactive group is brought into proximitywith a compatible reactive group on the target molecule. In a preferredembodiment, the reactive group is a photoactivatable group such that thegroup is only converted to a reactive species after illumination with anappropriate wavelength. An appropriate wavelength is generally a UVwavelength that is less than 400 nm. This method provides for specificattachment to only the target molecules, either in solution orimmobilized on a solid or semi-solid matrix.

Thus, a preferred method for specifically covalently labelingphosphorylated target molecules in a sample comprises:

-   -   i) contacting said sample with a binding solution comprising a        phosphate binding compound that is covalently bonded to a label        and a photoactivatable chemically reactive group; a salt        comprising trivalent metal ions and an acid;    -   ii) incubating said sample and said binding solution for        sufficient time to allow said phosphate binding compound and        said metal salt to associate with said phosphorylated target        molecule;    -   iii) illuminating said phosphate binding compound with an        appropriate wavelength where said photoactivatable reactive        group specifically forms a covalent bond with said        phosphorylated target molecule whereby said phosphorylated        target molecule is labeled.

This method is particularly useful when a stronger association is neededthan can be obtained with the non-covalent binding of the presentternary complex. In certain aspects, it is desirable that after theformation of the ternary complex that the pH be raised to about 7.0instead of an acidic pH. This method facilitates that process due to thestable formation of the covalent bond between the chelating moiety andthe target molecule. In addition, a covalent bond is also desirable forisolation purposes wherein perturbation of the system may lead tounstable ternary complexes. Preferred compounds for covalently labelingphosphorylated target molecules includes Compounds 34, 36, 39, 42 and44.

Unexpectedly, phosphorylated target molecules, typically peptides, canalso be isolated from a complex solution by taking advantage of theinsoluble nature of the ternary complex when higher concentrations ofthe more hydrophobic phosphate-binding compounds, such as Compound 5,are used in a moderately acidic environment with essentially equimolarmetal ion concentrations.

Methods of the present invention for isolating phosphorylated targetmolecules from solution comprise the following steps:

-   -   i) contacting the sample with a binding solution of the present        invention;    -   ii) incubating the sample of step i) and the binding solution        for sufficient time to allow said compound to associate with the        phosphorylated target molecule to form a ternary complex; and,    -   iii) separating said complex from said sample, whereby said        phosphorylated target molecules are isolated.

Hydrophobic phosphate-binding compounds of the invention when present ina binding solution at a concentration up to a hundred times higher thana binding solution for detection purposes typically form insolubleaggregates when the ternary complex forms. This property of the certainhydrophobic phosphate-binding compounds was taken advantage of todevelop a method for isolation of phosphopeptides. Thus, when a bindingsolution comprising certain hydrophobic phosphate-binding compounds isincubated with a sample in a way to facilitate formation of the ternarycomplex, the complex can be precipitated out of solution bycentrifugation (Example 13). Therefore, typically the binding mixtureand sample solution is vortexed, or mixed in a manner well known tothose skilled in the art, to simultaneously facilitate binding(formation of aggregates) and prevent precipitation of the ternarycomplexes. Following formation of the ternary complex, the solution istreated in such a way as to isolate the precipitated complexes, whereina preferred method is centrifugation. The resulting pellet comprisesphosphorylated target molecules that can be further analyzed, by methodssuch as MS. This method takes advantage of the affinity “pull-down” ofphosphopeptides or phosphoproteins from a complex solution (e.g., a cellextract protein digest), whereby at an acidic pH phosphate-bindingcompounds can complex with metal (typically gallium) ions and thephosphopeptides or phosphoproteins to form a precipitate. In addition,for the methods used to precipitate phosphorylated target molecules fromsolution, aluminum ions and ferric chloride comprising iron ions can bealso used for the formation of the ternary complex.

The present invention also contemplates further isolation, afteraggregated, of the phosphorylated target molecules, wherein thephosphate-binding compounds are removed from the phosphorylated targetcompounds, resulting in a solution free of phosphate-binding compounds.This is accomplished when the phosphate-binding compounds optionallycomprises a tag label such as a hapten, wherein the tag label functionsas a handle by which the phosphate-binding compounds can be pulled awayfrom the phosphorylated target molecules. A preferred tag label isbiotin wherein a matrix comprising biotin-binding proteins would be usedas the medium to separate the phosphate-binding compounds from thephosphorylated target molecules. Specifically, the resultingprecipitation pellet is resuspended in a solvent that disassociates themetal ion, phosphate-binding compound and phosphorylated target moleculecomplex, such as a basic solution, about pH 7-10, or through use of achelator such as EDTA or EGTA. The solution is then added to a matrix,such as a column containing Sepharose beads bound to a biotin-bindingprotein, wherein the phosphate-binding compounds comprising biotin bindto the beads and the phosphorylated target molecules pass through thecolumn. The resulting eluant contains phosphorylated target moleculesfree from phosphate-binding compounds that may be desirable for certainapplications. Alternatively, the dissociated mixture ofphosphate-binding compounds, metal ions and phosphorylated targetmolecules can be incubated with beads comprising biotin-binding proteinsas a slurry, wherein removal of the beads by gravity, such as by sizeexclusion or centrifugation, results in a solution of phosphorylatedtarget molecules without phosphate-binding compounds. Preferredcompounds for the formation of a precipitable ternary complex includecompounds 2, 5, 9, 12, 20, 21 and 22.

In some cases, phosphate-binding compounds can be removed fromphosphorylated target molecules without the need for affinitypurification. In this way, the aggregated ternary complex is contactedwith an organic extraction buffer (Example 27). Mixing of the pelletwith an organic solvent, such as acetonitrile, chloroform and waterresults in the phosphorylated peptide entering in the aqueous phase andthe phosphate-binding compound dissolving in the organic phase.

The isolated phosphorylated target molecules can be analyzed by a numberof methods, including but not limited to, gel electrophoresis, MALDI-TOFMS, or LC-MS/MS. Additionally, as described below, the phosphopeptidescan be derivatized using β-elimination, with subsequent addition ofnucleophiles to aid in identification of the site of phosphorylation.

In addition to isolation of phosphorylated target molecules from acomplex sample in solution, the present invention also contemplates theisolation of target molecules by capturing the phosphorylated targetmolecules using immobilized phosphate-binding compounds (Example 15, 25and 26). This can be done in a number of ways and the method isexemplified using an affinity column, ferrofluid beads and membranes;however, the methods illustrated are not intended to be a limitation ofthe method.

The methods of the present invention for isolating phosphorylated targetmolecules from solution using immobilized phosphate-binding compoundstypically comprise the following steps:

-   -   i) charging a matrix comprising an immobilized phosphate-binding        compound, wherein a metal-chelating moiety comprises Formula IV,        with a salt comprising metal ions;    -   ii) equilibrating the matrix with a moderately acidic binding        buffer,    -   iii) adding the sample to the matrix, wherein the phosphorylated        target molecules are bound to the matrix of step ii); and    -   iv) eluting the phosphorylated target molecules from the matrix,        whereby said phosphorylated target molecules are isolated.

The matrix can be any matrix known to one skilled in the art, includingpolymeric membranes, polymeric particles such as agarose, latex,magnetic or Sepharose beads, and glass, such as slides, beads or opticalfibers. The beads can be present in slurry or as a packed column throughwhich the sample passes and the membranes capture the phosphorylatedtarget molecules. An example of such a column is an affinity matrixcomprising phosphate-binding compounds bound to agarose (for instance,Compounds 13 and 14) or a resin (immobilized affinity column (IMAC)).Other compounds that find use in this method include, among others,Compounds 15, 20, 21 and 22.

Unlike the precipitation method, where an affinity column or organicextraction buffer can be used to remove phosphate-binding compounds fromthe isolated phosphorylated target molecules, the matrix in this methodcan optionally comprise just the metal-chelating moiety component of thephosphate-binding compound, which is subsequently bound with metal(preferably gallium) ions following the addition of the metal salt.However, a phosphate-binding compound represented by formula (A)m(L)n(B)can form the matrix wherein A is a reactive group that is used to attachB by way of L to the matrix material. Thus, the matrix is charged withthe metal ion, prior to addition of the sample. The matrix is thenequilibrated with a moderately acidic binding buffer; alternatively, themetal ion and acidic binding buffer would be present in one solution.The acidic binding buffer typically uses the same components as thebinding solution. A sample in an acidic binding buffer is then added tothe mixture, where phosphorylated target molecules will bind the metalions complexed to the metal-chelating moiety. Isolation of thephosphorylated target molecules is accomplished by an addition of asolution, which dissociates the ternary complex of the phosphate-bindingcompound (metal-chelating moiety), metal ion and phosphorylated targetmolecules. Preferably, the elution solution comprises a base and a basicpH-buffering agent. Useful bases include, without limitation, bariumhydroxide, sodium hydroxide and ammonium hydroxide. Alternatively, basicamine solutions are also useful elution agents. Any base that iscompatible with the sample and metal ion phosphorylated target moleculecomplex that dissociates the complex is preferred. In addition, organicsolvents such as acetonitrile is useful in eluting phosphorylated targetmolecules from the phosphate-binding compound matrix, and may bepreferable, depending on the subsequent analysis of the phosphorylatedtarget molecules, such as with MS.

As many phosphorylated target molecules often exist only in lowabundance, the isolation methods of the present invention are especiallyuseful for the purification and enrichment of such phosphorylated targetmolecules. These methods are useful for purifying phosphorylatedpeptides from crude peptide mixtures, which is advantageous for methodsthat subsequently analyze the peptides by MALDI, MS or nanoelectrospraytandem mass spectrometry (MS/MS). It is contemplated that a wide varietyof methods can be used to prepare samples purified and/or enriched bythe affinity matrix or separated from a complex solution. For example,dried separated phosphopeptides can be resuspended in water for LC-MSanalysis.

The IMAC of the present invention is also readily adaptable tomicrofluidics applications, such as the CD technology developed by GyrosAB (Uppsula, Sweden), wherein high-throughput screening of samples forproteomic analysis, such as peptide mapping with MALDI-TOF, can beaccomplished. Briefly, the Gyros AB technology comprises a CDmicrolaboratory with hundreds of microstructures (columns), whereinsamples are run through the columns based on centrifugation speeds andthe eluted sample is analyzed on the CD, permitting the entire processfrom a protein digest to MS analysis to be conducted on the CD. Thecolumns can be packed with particles that comprise BAPTA compounds(Compounds 13 or 14); samples can be then run through the columns andeither analyzed for phosphorylated peptide concentration, by fluorescentor chemiluminescent signal, or applied to a matrix on the CD forMALDI-TOF analysis. Thus, the methods of the present invention areamenable to microfluidics for high-throughput screening of samples,which is advantageous for proteomic studies.

Thus, the invention provides analytical reagents and methods for usewith mass spectrometry-based methods for the rapid, and quantitativeanalysis of phosphoproteins or phosphopeptides in a mixture. Thereagents and methods can be applied to the detection and identificationof proteins in sample mixtures of proteins, where the peptides isolatedby the method are characteristic of the presence of a protein in themixture. Isolated peptides or proteins can be characterized by massspectrometric (MS) techniques, and by application of sequence databasesearching techniques for identifying the protein from which thesequenced peptide originates.

The following references are examples of mass spectrometric techniquesfor protein identification, and can be used with the materials andmethods of the present invention: Ideker et al., “Integrated Genomic andProteomic Analyses of a Systematically Perturbed Metabolic Network,”Science 292:929-934 (2001); Gygi & Aebersold, “Measuring Gene Expressionby Quantitative Proteome Analysis,” Curr. Opin. Biotechnol. 11:396-401(2000); Goodlett et al., “Protein Identification with a Single AccurateMass of a Cysteine-containing Peptide and Constrained DatabaseSearching,” Anal. Chem. 72:1112-8 (2000); Goodlett et al., “QuantitativeIn Vitro Kinase Reaction as a Guide for Phosphoprotein Analysis by MassSpectrometry,” Rapid. Commun. Mass. Spectrom. 14:344-8 (2000); McLachlin& Chait, “Analysis of Phosphorylated Proteins and Peptides by MassSpectrometry,” Current Opin. Chem. Biol. 5:591-602 (2001); Aebersold &Goodlett, “Mass Spectrometry in Proteomics,” Chem. Rev. 101:269-295(2001); Vener et al., “Mass Spectrometric Resolution of ReversibleProtein Phosphorylation in Photosynthetic Membranes of Arabidopsisthaliana,” J. Biol. Chem. 276:6959-66 (2001); Zhou et al., NatureBiotechnol. 19:375-8 (2001). Those of skill in the art will recognizethese currently available mass spectrometry methods as compatible withthe materials and methods of the present invention. However, the presentinvention also contemplates that the materials and methods can be usedwith mass spectrometry techniques yet to become available that achievethe same or similar results. In addition, it is contemplated by thepresent invention that, prior to detection, phosphopeptides can besubjected to reverse phase, normal phase or ion-exchange columns toremove undesired materials from the phosphopeptide sample.

The present invention also contemplates alternative methods ofdetection, purification and/or enrichment. For example, the materialsand methods of the present invention may be used with Luminextechnology, which involves the labeling of latex microbeads with twofluorophores (U.S. Pat. No. 5,981,180 and U.S. Pat. No. 6,268,222).Using precise ratios of the two fluorophores, many different bead setscan be created, each one being unique and distinguishable in a laserbeam, based on the color code that results from the ratio of the twodyes. Instead of a capture antibody for a specific molecule coupled to aspecific bead set, the metal-chelators of the present invention may beused, i.e., phosphate-binding compound and metal ion complex orbiotin-binding protein that would bind biotin-labeled phosphate-bindingcompounds. However, antibodies that bind the sample could be used andthe phosphorylated target molecule could be detected with the bindingsolution of the present invention. For example, after an analyte isbound to the metal-chelator complex on the bead, a detector antibodycoupled to phycoerythrin may be used as a reporter. The end result is anantibody/metal-chelator sandwich assay on the color-coded microbead. Thebeads and the reporter molecule may be read on a Luminex 100 instrumentusing a dual laser system as they pass through a flow cell. One laserdetects the beads (the color code for an assay) and the other laserdetects the reporter signals. Thus, it is contemplated by the presentinvention that instead of a detector antibody, the metal-chelatingcomplex may be used in accordance with the bead detection for theseparation and detection of phosphorylated target molecules.

In the alternative, magnetic bead separation for automated bead andparticle capture systems, for example, LifeSep magnetic beads by DexterMagnetic Technologies or Captivate ferrofluid (Molecular Probes, Inc),may be used with the materials and methods of the present invention(U.S. Pat. No. 6,413,420; U.S. Ser. No. 08/868,598; US Application20020117451; U.S. Pat. No. 4,339,337; and U.S. Pat. No. 5,834,121) orferrofluid beads (Example 27). Magnetic separation works by means ofspecific affinity coatings attached to tiny magnetic beads, such as thephosphate-binding compounds. Beads are mixed with a sample containingphosphorylated target molecules such that the phosphorylated targetmolecules have the opportunity to bind tightly to the metalion/phosphate-binding compound on the bead. Once attached, the bead andthe ternary complex can be separated using a strong magnetic field.Depending on the process, the phosphorylated target molecule may eitherbe left bound to the bead or released by washing in a suitable solventor a basic buffer. Thus, efficient and rapid isolation is possible and,therefore, it is contemplated by the present invention, that thephosphate-binding compound/metal ion complex may be used with well-knownmethods of magnetic bead separation.

Thus, a wide variety of materials and methods are provided for theseparation, purification and enrichment of phosphorylated targetmolecules, including the novel use of an immobilized affinity matrix.

The present invention provides compounds and methods for thedifferential isolation and identification of phosphorylated serine,threonine or tyrosine amino acids. The materials and methods describedabove for the labeling and isolation of phosphorylated target molecules,absent mass spectrometry or other similar techniques, are generally usedfor detecting protein phosphorylation, but do not give information onthe specific location of the phosphate on the protein or polypeptide.The present invention contemplates further analyzing isolatedphosphorylated proteins or peptides obtained by immobilized affinitymatrix or precipitation methods described above to differentiallyidentify phosphorylated peptides. Isolated phosphorylated proteins aresubjected to proteolytic digestion, followed by acid hydrolysis oralkaline hydrolysis and analyzed.

A base such as barium hydroxide or sodium hydroxide catalyzes thedephosphorylation of the peptides, forming activated dehydroalaninederivatives, which are vulnerable to attack by amine or thiol-containingcompounds, resulting in the formation of stable derivatives of theoriginal phosphopeptide. These derivatives are more hydrophobic and aretherefore more amenable to identification by HPLC, mass spectrometry, orby Edman sequencing. Under the conditions used, phosphoserine residuesundergo elimination and addition, phosphothreonine residues undergoelimination but not addition and phosphotyrosine residues are unalteredby the treatment. Thus, differential identification can be accomplishedbased on this knowledge. In Edman degradation, during the acid or basedelivery the phosphate is β-eliminated and the resulting dehydro-aminoacids rapidly form a dithiothreitol (DTT) adduct. See Meyer et al.,FASEB J. 7:776 (1993). In contrast, O-Hex-N-Ac on serine and threonineis stable in Edman degradation. See Gooley & Williams, Nature 358:557(1997). Thus, the present invention may be used to differentiate betweenserine or threonine phosphorylation and glycosylation.

Edman degradation is thus an effective method for quantitating serineand threonine, following β-elimination and derivatization. See Yan etal., “Protein Phosphorylation: Technologies for the Identification ofPhosphoamino Acids”, J. Chromatogr. 808:23-41 (1998)). These modifiedproducts also survive acid hydrolysis, and can be quantitated byreversed-phase HPLC analysis. See, e.g., Meyer et al., J. Chromatogr.397:113 (1987) and Holmes, FEBS Lett. 215:21 (1987). Using a similarapproach, characterization by capillary zone electrophoresis andlaser-induced fluorescence has also been used to quantitate thephosphoserine content of peptides and proteins. See, Fadden & Haystead,Anal. Biochem. 225:81 (1995).

Nanoelectrospray MS/MS is used for phosphopeptide sequencing for exactdetermination of phosphorylation sites. See Stensballe et al.,“Characterization of Phosphoproteins From Electrophoretic Gels byNanoscale Fe(III) Affinity Chromatography With Off-Line MassSpectrometry Analysis,” Proteomics 1:207-222 (2001). In-gel digestionscan be achieved as described in Shevchenko et al., Anal. Chem. 68:850-58(1996) and Jensen et al., Meth. Mol. Biol. 112:513-30 (1998). Thepresent invention also contemplates that the materials and methods canbe used with mass spectrometry techniques yet to become available thatachieve the same results.

The sequence of phosphopeptides and the identification of the site(s) ofphosphorylation can also be determined by a combination of tandem massspectrometry and computer-assisted database search programs, such asSEQUEST (Trademark, University of Washington, Seattle Wash.) (McCormacket al., “Direct Analysis and Identification of Proteins in Mixtures byLC/MS/1\4S and Database Searching at the Low-Femtomole Level,” Anal.Chem. 69:767-776 (1996); Eng et al., “An Approach to Correlate TandemMass Spectral Data of Peptides with Amino Acid Sequences in a ProteinDatabase,” J. Amer. Soc. Mass. Spectrom. 5:976-989 (1994); U.S. Pat. No.5,538,897. While a variety of MS methods are available and may be usedin these methods, MALDI/MS and Electrospray Ionization MS (ESI/MS)methods are typically used.

Sample Preparation

The sample is defined to include any material that may containphosphorylated target molecules, substrates that interact with kinasesand phosphatases, substances that interact with kinase and phosphatasesubstrates and any substance that binds phosphorylated target molecules.Typically the sample is biological in origin and comprises tissue, acell or a population of cells, cell extracts, cell homogenates, purifiedor reconstituted proteins, recombinant proteins, fusion proteins, bodilyand other biological fluids, viruses or viral particles, prions,subcellular components, or synthesized peptides or proteins. Possiblesources of cellular material used to prepare the sample of the inventioninclude, without limitation, plants, animals, fungi, bacteria, archae,or cell lines derived from such organisms. The sample can be abiological fluid such as whole blood, plasma, serum, nasal secretions,sputum, saliva, urine, sweat, transdermal exudates, cerebrospinal fluid,or the like. Alternatively, the sample may be whole organs, tissue orcells from an animal. Examples of sources of such samples includemuscle, eye, skin, gonads, lymph nodes, heart, brain, lung, liver,kidney, spleen, solid tumors, macrophages, mesothelium, and the like.

Prior to combination with the binding solution of the present invention,the sample is prepared in a way that makes the phosphorylated targetmolecules or enzyme substrates in the sample accessible to thephosphate-binding compounds. Alternatively, the sample may compriseenzymes or binding proteins that interact with phosphorylated targetmolecules. Typically, the samples used in the invention comprise tissue,cells, cell extracts, cell homogenates, purified or reconstitutedproteins, peptides, recombinant proteins, biological fluids, lipids,amino acids, nucleic acids and carbohydrates or synthesized proteins.However, the desired target (target molecule comprising exposedphosphate groups) may require purification or separation prior toaddition of the binding solution due to the presence of other discretebiological components. The desired phosphorylated target molecules andother discrete biological components can be optionally separated fromeach other or from other components in the sample by mobility (e.g.,electrophoretic gel or capillary) by size (e.g., centrifugation,pelleting or density gradient), or by binding affinity (e.g., to afilter membrane or affinity resin) in the course of the present methods.For example, when the sample is to be separated on an SDS-polyacrylamidegel, the sample is first equilibrated in an appropriate buffer, such asan SDS-sample buffer containing Tris, glycerol, DTT, SDS, andbromophenol blue. For certain aspects of the invention it is preferredthat the phosphorylated target molecules not be separated beforeanalysis.

When starting with a sample source that is not appropriate forseparation, e.g., whole cells or tissue homogenate, the sample needs tofirst be prepared, using techniques well known to those skilled in theart. Preparation of the sample will depend on how the phosphorylatedtarget molecules are contained in the sample (see e.g., CurrentProtocols in Molecular Biology; Herbert, Electrophoresis 20:660-663(1999)). For example, an optional way of preparing samples for 2-D gelelectrophoresis followed by labeling with the compositions and methodsof the present invention includes lysing cells using a lysis buffer thatensures that the proteome, in addition to post-translationalmodifications, of a sample remain in their in vivo state throughout theentire procedure. Examples of such buffers include ones derived from aurea/NP-40/2-mercaptoethanol mixture. Therefore, the lysis buffer mightadditionally contain phosphatase inhibitors such as sodiumorthovanadate, sodium fluoride or β-glycerophosphate in addition to aprotease inhibitor cocktail.

Typically the phosphorylated peptides and proteins in the sample have amolecular weight greater than about 500 daltons. More typically thephosphorylated peptides and proteins are more than 800 daltons. In oneaspect of the invention, the phosphorylated proteins comprise a mixtureof phosphorylated proteins with different molecular weights that fallwithin a range of molecular weights, wherein the phosphorylated proteinsare used as molecular weight standards so that labeled phosphorylatedproteins or peptides can be accurately analyzed. Samples comprisingphosphorylated peptides subjected to the methods of the presentinvention can be generated from natural or synthetic samples and may bethe result of chemical, physical or enzymatic digestion ofphosphorylated protein samples. Proteins can be digested using anyappropriate enzymatic method, such as trypsin digestion. Peptides in thedigest may be preferably sized to facilitate peptide sequencing usingtandem mass spectrometric methods, and are typically in the size rangefrom about 10 to about 50 amino acids in length. Alternatively, thesepeptides can also function as phosphatase substrates in a method of theinvention to identify such enzymes and to measure their quantity and/orenzymatic activity.

Samples comprising phospholipids, wherein the phospholipids are thetarget molecules, are prepared with modifications compared to samplescomprising phosphoproteins prior to applying to solid or semi-solidmatrix due to their hydrophobic nature. Most samples typically requiresome sort of extraction treatment prior to binding with the compositionsand methods of the present invention. Where the phosphorylated targetmolecule of interest come from tissue samples or samples from organismshaving cell walls, mechanical or chemical disruption may be required.Suitable means are well known in the art and include, but are notlimited to, the use of a tissue homogenizer or a French pressure cell inconjunction with, for example, organic solvent extractions. Methods ofcell disruption and fractionation are described in Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons 1997). Samplesmay be extracted with solvents possessing varying hydrophobicproperties, and the optimal solvent is contingent upon the nature of thephosphorylated target molecule of interest. Extraction techniquesconventional in the art that result in a sample suitable for detectionare contemplated by the present invention.

The following references describe various extraction techniques: Dole etal., J. Clin. Invest. 35:150-54 (1956); Dole et al., J. Biol. Chem.235:2595-99 (1960); Bligh et al., Canadian J. Biochem. Physiol.37:914-17 (1959); Folch et al., J. Biochem. 226:497-509 (1957). The Doleet al. references describe an extraction method that involvesextractions of the sample with an isopropyl alcohol/heptane/sulfuricacid mixture, followed by several heptane extractions. The organic phaseis dried with nitrogen for use in subsequent steps. The Folch et al.reference describes the extraction of lipids from biological tissuehomogenates or body fluids. Samples are extracted withchloroform/methanol, filtered and reverse-extracted with 0.1 M KCl. TheBligh et al. reference describes the organic extraction of lipids frombiological tissue homogenates or fluids. Samples are extracted withmethanol/chloroform and chloroform, and then filtered andreverse-extracted with water.

Typically, the phosphorylated target molecules (proteins, peptides,carbohydrates or lipids) are present on or in a solid or semi-solidmatrix. In one aspect of the invention, this matrix comprises anelectrophoresis medium, such as a polyacrylamide gel, agarose gel,linear polyacrylamide solution, polyvinyl alcohol gel or a hydrogel. Thesolid or semi-solid matrix can also comprise a membrane, such as afilter membrane, a nitrocellulose, poly(vinylidene difluoride) (PVDF)membrane, or nylon membrane wherein the phosphorylated target moleculesare immobilized on the membrane by blotting, spotting, electroblotting(tank and semi-dry), capillary blotting or other methods of applicationwell known to those skilled in the art. In accordance with the presentinvention, a solid and semisolid matrix also includes a glass slide, aplastic matrix (e.g., multi-well plate or pin), a glass or polymericbead or fiber or a semiconductor material. The phosphorylated targetmolecules may be arrayed on the support in a regular pattern orrandomly. A preferred array of the present invention is a hydrogel glassslide support, wherein the phosphorylated target molecules of the sampleare arrayed in a regular pattern. The present invention contemplatesthat the phosphorylated target molecules can be phosphorylated afterimmobilization on a matrix material, wherein an enzyme substrate isimmobilized and the appropriate enzyme and phosphate is incubated withthe immobilized substrate. For certain aspects of the invention it ispreferred that the phosphorylated target molecules be free from a solidor semi-solid matrix, i.e. not immobilized and present in an aqueoussolution as solubilized molecules.

Illumination

In a typical detection method, at any time after or during binding withthe phosphate-binding compounds of the present invention, the sample isvisualized whereby the phosphorylated target molecule is detected.Visualization can comprise different methods and is dependent on thechemical moiety A that is covalently attached to the metal-chelatingmoiety of the phosphate-binding compound. When the chemical moiety A isa label, visualization typically comprises illumination with awavelength of light capable of exciting the reagent to produce adetectable optical response, as defined above, and observed with a meansfor detecting the optical response. Equipment that is useful forilluminating the phosphate-binding compounds of the invention includes,but is not limited to, hand-held ultraviolet lamps, mercury arc lamps,xenon lamps, lasers and laser diodes. These illumination sources areoptionally integrated into laser scanners, fluorescence-based microplatereaders, standard or minifluorometers, or chromatographic detectors. Thedegree and/or location of binding, compared with a standard or expectedresponse, indicates whether and to what degree the sample possesses agiven characteristic, i.e., phosphorylated target molecules.

The optical response is optionally detected by visual inspection, or byuse of any of the following devices: CCD cameras, video cameras,photographic film, laser-scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,fluorescence-based microplate readers, or by a means for amplifying thesignal such as photomultiplier tubes.

The detectable optical response can be quantified and used to measurethe degree of phosphorylation of the phosphorylated target molecule inthe sample mixture. Quantification is typically performed by comparisonof the optical response to a prepared standard or to a calibrationcurve. Typically, the measured optical response is compared with thatobtained from a standard dilution of a known concentration of thephosphorylated target molecule in an electrophoretic gel, HydroGel or ona membrane. Generally, a standard curve must be prepared whenever anaccurate measurement is desired. Alternatively, the standard curve isgenerated by comparison with a reference dye or dyed particle that hasbeen standardized versus the reagent-target conjugate desired.

Alternatively, stained electrophoretic gels are used to analyze thecomposition of complex sample mixtures and additionally to determine therelative amount of a particular phosphorylated target molecule insamples. This can be accomplished, for instance, in conjunction withdetermination of the number of phosphate groups on a molecule and atotal protein stain to differentiate between an increase in the amountof protein versus an increase in phosphate groups on a particularprotein or peptide.

Inductively coupled plasma mass spectrometry (ICP-MS) is a usefultechnology for the trace elemental analysis of environmental,biological, and pharmaceutical samples. Recently, the feasibility ofdirectly measuring phosphorous as m/z 31 signal liberated from β-caseinusing laser ablation ICP-MS has been demonstrated on electroblotmembranes (Marshall, P., et al. Analyst 127: 459-461 (2002)). Though 16pmole of the pentaphosphorylated protein was detectable on blots, thetechnique was not successfully performed on polyacrylamide gels due tovery high background signal. This was undoubtedly due to the presence ofisobaric species and overlap from adjacent species generated from thepolyacrylamide gel matrix and electrophoresis buffer components. Thedetection of low concentrations of phosphorous presents severalanalytical challenges for ICP-MS due to its poor ionization in the argonICP and the presence of interfering polyatomic species directly at mass31 (¹⁵N¹⁶O and ¹⁴N¹⁶O¹H) and indirectly at mass 32 (¹⁶O₂ and ³²S).ICP-MS could be used to detect phosphoproteins stained with the methodsof this invention. The detection procedure is envisioned to involve thefollowing steps. First, proteins separated by gel electrophoresis arefixed to remove the SDS. A typical fixative would be 40% methanol/10%acetic acid. Next, gels would be stained for phosphoproteins using themethods of the invention. Next, the gels would be washed to removeexcess stain. The more prominent phosphoproteins could be visualized byfluorescence imaging at this point and background staining can beminimized by inspection and adjustment of wash times as appropriate.Gels are then dried down and the gel is subjected to laser ablationICP-MS by methods similar to those described in Marshall et al, 2002.Sampling can be performed by single or multi-spot analysis,straight-line scans or rastering. In the case of rastering, virtual gelscan be constructed from the data obtained as described by Loo R R, etal., Anal Chem. 73:4063-70 (2001)). Using the ruthenium-containingSYPRO® Ruby dye staining technology, gallium (aluminum or iron) signalfrom the phosphoprotein stain, as well as ruthenium for the totalprotein stain could be independently quantified.

Thus, it is contemplated by the present invention that a wide variety ofinstrumentation may be used to detect the phosphorylated targetmolecules, e.g., electrospray ionization (ESI) tandem mass spectrometry(MS/MS). A series of different techniques, including automated highperformance liquid chromatography (HPLC)-MS/MS, capillary-HPLC-MS/MS,and solid phase extraction (SPE)-capillary zone electrophoresis(CZE)-MS/MS, are described in Figeys et al., Electrophoresis19:1811-1818 (1998).

When measuring fluorescence polarization, many forms of automation maybe used and are known by those skilled in the art. As one example, anystandard fluorometer equipped for polarization experiments ormeasurements may be used in practicing this embodiment of the inventionto both irradiate the mixture and measure the polarization. Wavelengthssuitable to excite the fluorophore depend on the nature of thefluorophore, as described above. Typically, one uses cut-off filters todefine a wavelength range, which is determined by the excitation andemission wavelengths of the fluorophore. For example, forfluorescein-labeled peptides, one would typically use an excitationcutoff filter of 485 nm. Standard fluorometers can be used, or, forexample, a fluorescence-based plate reader. Thus, in addition, one ofskill in the art of automation may use various instruments to measurefluorescence polarization in accordance with the materials and methodsof the invention.

The use of1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid(TF-BAPTA) or any of the fluoride-containing phosphate-binding moleculesdescribed in this invention with gallium for binding to thephosphorylated target molecule could be detected using 19_(F)-NMRspectroscopy (Doughty D A, Tomutsa L. Magn Reson Imaging 1996;14(7-8):869-73). Additionally, radioactive gallium-67 (half-life: 78hr), gallium-68 (half-life: 1.13 hr) or gallium-72 (half-life: 14.1 hr)could be employed with any of the phosphate-binding compounds of theinvention to generate a detectable signal by autoradiography orscintigraphy.

As described above, while a wide variety of methods of detection areused with the present invention, a preferred method includes the use offluorescence. Fluorescence from the phosphate-binding compound metal(preferably gallium) ion complex simultaneously binding to thephosphorylated target molecule can be visualized with a variety ofimaging techniques, including ordinary light or fluorescence microscopy.

Kits

Suitable kits for labeling, isolating and identifying enzymes thatinteract with phosphorylated target molecules also form part of theinvention. Such kits can be prepared from readily available materialsand reagents and can come in a variety of embodiments. The contents ofthe kit will depend on the design of the assay protocol or reagent fordetection or measurement. All kits will contain instructions,appropriate reagents and phosphate-binding compounds, separation media,and solid supports, as needed. Typically, instructions include atangible expression describing the reagent concentration or at least oneassay method parameter such as the relative amounts of reagent andsample to be added together, maintenance time periods for reagent/sampleadmixtures, temperature, buffer conditions and the like to allow theuser to carry out any one of the methods or preparations describedabove.

A kit for binding phosphorylated target molecules comprises a bindingreagent that is typically prepared in solution, wherein the bindingsolution is identical to what was described above. Optionally, the kitwould comprise any one of the following; molecular weight markers forboth phosphorylated and non-phosphorylated target molecules, a totalprotein stain and a staining solution for glycoproteins. When the kit isused to detect phosphorylated proteins or peptides in a gel or on ablot, molecular weight markers are typically part of the kit.Alternatively, when the kit is used to stain phosphorylated targetmolecules in a solution or on an array, molecular weight markers wouldtypically not be part of the kit.

Another kit in the present invention finds use in identifying kinases orphosphatases or measuring their activity and/or evaluating the effect ofinhibitors and activators on these enzymes. Typically this kit wouldcomprise appropriate substrate immobilized on matrix material, bindingsolution and appropriate controls. Alternatively the phosphate-bindingcompounds would be immobilized on the matrix and the end user wouldsupply both substrate and enzyme. This kit would comprise a solutioncontaining a metal ion salt and an acid or appropriate buffer that whenadded to the immobilized phosphate-binding compounds would form thebinding solution of the present invention.

Another kit of the present invention finds use in isolatingphosphorylated proteins or peptides from a complex sample mixture,wherein the ternary complex is pulled out of solution. The kit wouldoptionally comprise a binding solution, elution buffer and optionally aprotein or hapten-binding support, wherein the support could be amultiwell plate, agarose resin, polymeric microbeads or magnetic beads,containing an appropriate affinity reagent, e.g. a biotin- orhapten-binding protein, an antibody, a lectin, a protein-binding nucleicacid or other biopolymer covalently attached to the support. The kitwould further optionally contain one or more of a spin column, standardpeptide mixture and nucleophilic derivatization compound.

Another kit of the invention that finds use in isolating phosphorylatedproteins or peptides from a complex sample mixture comprises a matrixcontaining phosphate-binding compounds that are covalently attached tothe matrix, typically in the form of a column. The kit would typicallycomprise, in addition to the phosphate-binding compounds immobilized onthe matrix, a metal salt, a wash buffer, a moderately acidic bindingbuffer, and an elution buffer. The metal salt is preferably galliumchloride and the elution buffer preferably comprises barium hydroxide.

Those skilled in the art will appreciate that a wide variety ofadditional kits and kit components can be prepared according to thepresent invention, depending upon the intended user of the kit, and theparticular needs of the user.

Applications

The present invention is useful for a wide variety of applications in awide variety of areas including, but not limited to, basic researchapplications, high-throughput screening, proteomics, microarraytechnology, diagnostics, and medical therapeutics. Those skilled in theart will appreciate that the invention can be used in a wide variety ofassay formats in a wide variety of diagnostic applications. Theforegoing description seeks merely to illustrate the many applicationsof the materials and methods of the present invention, and does not seekto limit the metes and bounds of the invention as described in the abovesections.

The materials and methods of the present invention are useful for anumber of applications. The present invention may be used to generatedata that are used as a reference point for a human patient or animalsample for the diagnosis of disease, progression of disease, and/orpredisposition for disease. By way of example, if a disease isassociated with changes in protein composition in certain cells, e.g.,protein phosphorylation in different organ systems, cell sources ortissue types, a patient sample may be used to generate a protein profileaccording to the materials and methods of the invention, and comparedwith profiles of corresponding samples of normal or non-diseased samplesand/or diseased origin to determine the presence or absence of,progression of, and/or predisposition to the particular disease inquestion. It is contemplated by the present invention that many diseasesmay be diagnosed with data or images generated by the materials andmethods of the present invention, including diseases for whichparticular aberrations in protein expression are either known or notknown. Such disease states include, but are not limited to, metabolicdiseases that are associated with the lack of certain enzymes,proliferative diseases that are associated with aberrant expression ofcertain genes, e.g., oncogenes or tumor suppressors, or developmentaldiseases that are associated with aberrant gene expression. Thus, if itis known that a given disease of interest is associated with certainchanges of a particular type of cell, tissue, cell source, or organsystem, a human patient or animal may be diagnosed simply based on itsindividual expression profile generated by 2-D gel electrophoresis oranother appropriate separation and analysis technique such as bead-basedanalysis technology developed by Luminex, or others, or evaluations doneon microarrays in accordance with any aspect of the present invention.In another aspect, expression profiles generated by one of these methodsmay be used to analyze a diseased organ, tissue or cell type andcompared with the corresponding profile counterpart obtained from anon-diseased sample.

Moreover, the information generated by the materials and methods of thepresent invention may be used to “backtrack” or identify and/orassociate novel or known genes and their corresponding products that areinvolved in the manifestation of, progression of, or predisposition to adisease of interest, and with the development of symptoms of aparticular disease, by generating the amino acid sequence of aphosphoprotein or phosphopeptide of interest based on the materials andmethods of the present invention. For example, ESTs are partialnucleotide sequences obtained from cDNA derived from mRNA from any givencell line. Thus, the present invention may be used to generate aminoacid sequence data, and from the amino acid sequence data, extrapolatepotential DNA sequences that can be used to search EST databases. Forexample, MS/MS sequence data in the form of a peptide sequence tag, maybe used to query EST databases if a protein is not identified bysearching the conventional full length sequence databases. If an EST isretrieved, then the corresponding DNA clone can be ordered andsequenced. The apoptotic protease FLICE/Caspase-8 and the trinucleotiderepeat binding protein p20-CGGBP was identified and cloned by thisapproach. See Muzio et al. “FLICE, a Novel FADD-homologousICE/CED-3-like Protease, is Recruited to the CD95 (Fas/APO-1)Death-Inducing Signaling Complex,” Cell 85:817-827 (1996) and Deissleret al., “Rapid Protein Sequencing by Tandem Mass Spectrometry and cDNACloning of p20-CGGBP. A Novel Protein that Binds to the Unstable TripletRepeat 5′-d(CGG)n-3′ in the Human FMR1 Gene,” J. Biol. Chem.272:16761-16768 (1997). During analysis of protein components isolatedfrom the human spliceosome, a relatively large number of ESTs wereretrieved by MS/MS data. In the alternative, it may be necessary togenerate amino acid sequence data for sequence homology searching, e.g.,by BLAST algorithm searching. If the sequence is significantly relatedto a characterized protein from another species, then its function maybe directly deduced. If no related proteins exist, however, then theamino acid sequence data may be used to design oligonucleotide probesfor cloning of the cognant gene. Complete sequence determination of theprotein can then be performed at the DNA level by established geneticand molecular biology techniques.

The phosphorylated target molecules can be from many different sources,including cell types, cell conditions, genetic background, states ofperturbation or of different developmental states. Cell sources foranalysis may be transgenic or non-transgenic, transfected ornon-transfected, virus- or prion-infected or non-infected.“Perturbation” refers to experimental manipulation of the sources, i.e.,cells, such as treatment with a particular compound or drug compared tonon-treatment of a drug. Alternatively, it can refer to treatment with aparticular compound or drug compared to treatment of a source or cellwith a different dosage of a particular compound or drug.

For example, cells can be subjected to a candidate drug regimen togenerate a phosphoprotein expression profile in accordance with thepresent invention. The images of 2-D gels generated in accordance withthe present invention may be stored on a database, and the database maybe later queried for a cell source representing a different treatment,e.g., protein expression profiles generated by a response to a differentdrug or where no drug is present, or where the candidate drug is used ina different way. Moreover, the candidate drug may bind specifically to aparticular protein, permitting analysis of cells or other sources, whichexpress that protein. The database query may derive information aboutcell sources that express a particular protein.

Thus, the materials and methods of the present invention could be usedto gain valuable information of the effects of various drugs andcompounds on the cellular phosphoprotein status. For example, it wasdemonstrated that the compounds FK-506, cyclosporin and rapamycin, usedto suppress tissue rejection, inhibit certain protein phosphatases.Schreiber et al., Cell 70:365-68 (1992). A database of lymphoid proteinsdetected by 2-D polyacrylamide gel electrophoresis has also beengenerated. The database contains 2-D patterns and derived informationpertaining to polypeptide constituents of unstimulated and stimulatedmature T cells and immature thymocytes, cultured T cells and cell linesthat have been manipulated by transfection with a variety of constructsor by treatment with specific agents, single cell-derived T and B cellclones, cells obtained from patients with lymphoproliferative disordersand leukemia, and a variety of other relevant cell populations. SeeHanash & Teichroew, “Mining the Human Proteome: Experience with theHuman Lymphoid Protein Database,” Electrophoresis 19:2004-2009 (1998).Thus, in accordance with the present invention, cells treated with asuspected drug compound can be compared to untreated cells to generate a2-D gel electrophoresis profile. Furthermore, it may be observed, forexample, that certain drug compounds induce the activation of differentsets of kinases or phosphatases. Such evidence could lead to theelucidation of the mechanism by which many drug compounds work andmanifest their effects.

A 2-D gel electrophoresis study was performed to generate aphosphoprotein profile in cultures that were subjected to the effect ofoxygen/glucose deprivation. The results suggested that this model couldbe a good method to observe the development of the tissue and itsresponse to an ischaemic lesion. See Tavares et al., “Profile ofPhosphoprotein Labeling in Organotypic Slice Cultures of RatHippocampus,” Neurochemistry 12:2705-2709 (2001).

The materials and methods of the present invention can also be used tostudy biological phenomena, such as, for example, signal transduction,mitosis, cell proliferation, cell motility, cell shape, gene regulation,and many other cellular processes. The mechanism of action of kinasesand phosphatases and the physiological relevance of site-specificphosphorylation of substrate proteins can be explored with the materialsand methods of the present invention. The materials and methods of thepresent invention offer the advantage of high-resolution 2-D gelelectrophoresis to simultaneously resolve hundreds of cellularpolypeptides. Using the materials and methods of the present invention,the potential for the identification of proteins and the expression oftheir genes at various stages of cell growth, differentiation, ordisease, is extensive. Thus, the invention provides methods andmaterials for the detection and quantitation of phosphorylation ofspecific cellular proteins that may provide insight into the mechanismsby which phosphorylation is employed for the regulation in cells.

It is well known that the critical events in the cell cycle arecontrolled by a complex interplay of kinases and phosphatases. Thus, thestatus of phosphorylation of different protein isoforms during differentphases of the life cycle is important to researchers. Thus, inaccordance with the materials and methods of the present invention, thephosphorylation of different proteins related to the stage of the cellcycle related to the activity of certain kinases or phosphatases may beexplored using the materials and methods of the present invention. Byway of example, a global analysis of phosphoproteins in cells can beused to analyze the primary signals of, for example, mitogenesis inselected cells, or in G1 or S phase cells. Thus, the materials andmethods of the present invention may be useful in investigating thephosphorylation status of various proteins during the cell cycle.

Those of skill in the art will recognize that a database can begenerated using the materials and methods of the present invention toproduce a record that may show the correlation between gene expressionat the RNA and protein level to the function of the cell. For examples,in situations where the cells under study are obtained in both cancerousand normal conditions, comparison of the relative gene expression can beused to identify genes that can serve either as diagnostic markers ofpathology or as sites for the pharmacological intervention or treatmentof, for example, cancer. Similarly, other diseases can be analyzedmerely by substituting the source of cells for analysis.

Thus, the present invention may be used to generate a comprehensivephosphoprotein expression profile from any cell type or biological fluidof interest. A cell type of interest may be any cell, or portion thereofwith genetic material. A reference cell can be of any cell type in whichthe difference in protein expression patterns and levels is desired tobe measured. Preferably, the cells are maintained as similar to theirnative state as possible and culture techniques, incubation times etc.,are performed identically between the two to minimize any non-naturallyoccurring differences. For example, development of a comprehensiveprotein profile of pre-cancerous, and/or malignant test cells and anormal reference cell can be achieved according to the invention. Suchexpression profiles can be used to characterize molecular events, forexample, related to tumor development and the cellular mechanismsinvolved.

In accordance with the present invention, a cell of interest and areference cell could be obtained from the same patient to get anindividual phosphoprotein expression profile that can be used todiagnose or treat that patient for those diseases that involve proteinphosphorylation. For example, when a tumor is excised, a margin ofnon-transformed cells is typically removed as well. Phosphoproteinexpression profiles can help to ensure that the cells removed all hadsimilar profiles to normal cells rather than the metastatic cells fromthe same patient for those cancers that involve, or are thought toinvolve, protein phosphorylation.

One example of cell lines that may be analyzed using the materials andmethods of the present invention includes human tumor cell lines. Forexample, human tumor cell lines representing a broad spectrum of humantumors and exhibiting acceptable properties and growth characteristicsmay be grown according to standard methods for cell line expansion,cryopreservation and/or characterization for use with the presentinvention. If phosphorylation is implicated in cellular aging, thematerials and methods of the present invention may be used to analyzetest and reference cells, i.e., to develop phosphoprotein expressionprofiles associated with aging, such as different stages of ontogenesis,for example, protein profiles of embryonic liver-derived hematopoieticstem cells. Thus, the invention contemplates a comparison of anydiseased state to a normal reference state.

In addition, studying the effects of various ligands added to cells canassess the effects of various agonists on the reversible phosphorylationon multiple cellular proteins. Thus, for example, the in vivo substratesof a kinase of interest could be determined by treating cells withsuspected substrates and comparing the resulting gel images of 2-Dseparated proteins with untreated controls. As an increasing number ofcytokines are being discovered and characterized, many or all of whichwill activate protein kinases or phosphatases as they manifest theireffects on target cells, the materials and methods of the presentinvention may be especially useful for exploring such mechanisms. Forexample, the identity of some of these proteins may suggest assays to beformulated for the location and characterization of kinases andphosphatases induced by lymphokines or cytokines and lead to a betterunderstanding of autoimmune diseases. Methods for identifyingphosphoproteins upregulated in response to the cytokines IL-2 or IFN-γwere described using both silver staining and Western blotting forprotein detection and identification. The silver-stained profile servedas a “fingerprint” for phosphorylation events that occur in response tocytokine treatment. See Stancato & Petricoin III, “Fingerprinting ofSignal Transduction Pathways Using a Combination of Anti-PhosphotyrosineImmunoprecipitations and Two-Dimensional Polyacrylamide GelElectrophoresis,” Electrophoresis 22:2120-2124 (2001).

The materials and methods of the present invention can also be used tomap kinase and phosphatase substrates in vitro. For example theidentification of substrates for various kinases can be determined byprocessing extracts from cells and allowing a purified kinase tophosphorylate its substrate proteins. One skilled in the art couldcompare all the cytosolic proteins as candidate substrates for thekinase under investigation to identify major substrates for a kinase ofinterest. Similar to in vitro assays for kinases, it is possible to usethe advantages offered by 2-D separation and assays on microarrays, inmultiwell plates, in microfluidics devices, on microbeads and usingother high-throughput assay technologies and the invention to isolateand characterize the phosphatases that catalyze the removal of phosphatefrom phosphorylated substrates. Thus, the activity of kinases andphosphatases responsible for phosphorylating and dephosphorylatingindividual proteins can be analyzed. See, e.g., Fruehling & Longnecker,“In Vitro Assays for the Detection of Protein Tyrosine Phosphorylationand Protein Tyrosine Kinase Activities,” Methods in Mol. Biol. 174(Ch.36):337-343 (2001).

The applications described herein are provided merely to illustrate awide variety of potential uses of the invention, and are in no wayintended to limit the scope of the invention. A detailed description ofthe invention having been provided above, the following examples aregiven for the purpose of illustrating the invention and shall not beconstrued as being a limitation on the scope of the invention or claims.

EXAMPLES

Generally, the nomenclature as used herein, and the laboratoryprocedures in cell culture, molecular genetics, and protein chemistrydescribed below are those well known and commonly employed in the art.Generally, enzymatic reactions and purification steps are performedaccording to the manufacturer's specifications. Units, prefixes, andsymbols may be denoted in their SI accepted form. Numeric ranges areinclusive of the number defining the range and include each integerwithin the defined range.

Example 1 Determination of BAPTA Selectivity for Gallium and GalliumIons for Phosphorylated Target Molecules and a Screening Method forPhosphate-Binding Compounds

(A) BAPTA with Trivalent Gallium Ions Selectively DetectsPhosphoproteins.

A comprehensive search of metal-chelating compounds was performed toidentify fluorescent reagents that when combined with a gallium salt(gallium chloride) would selectively detect phosphorylated targetmolecules (particularly phosphopeptides and phosphoproteins) in amixture of phosphorylated and nonphosphorylated target molecules. Thecompounds were tested in a fluorescence spectrophotometer for theirability to bind gallium (III) ion and selectively detect thephosphoprotein ovalbumin. Binding to gallium (III) ion was determined bya fluorescence increase of the same compound in the presence of up to 5μM gallium chloride in 75 mM NaOAc (pH 4.0) and 140 mM NaCl. Ovalbumindetection was also judged by a fluorescence increase; however, thecompounds were placed in a solution containing 75 mM NaOAc (pH 4.0), 140mM NaCl, 1-4 μM ovalbumin, and 0.5 μM gallium chloride. Selectivity ofphosphoprotein detection was evaluated by virtual elimination of thefluorescence increase in the presence of the same solution lackinggallium chloride. Using compound 1, a variety of metal ions, includingiron and gallium, were screened to determine which ion(s) were bestsuited for phosphoprotein detection. Metal ions were assayed for bindingto compound 1, phosphoprotein detection, and general protein staining bymonitoring a fluorescence increase at 530 nm in 75 mM NaOAc (pH 4.0),140 mM NaCl, 0.5-5 μM metal ion, with or without 4 μM ovalbumin or 1 μMlysozyme. Only trivalent cations that bound to compound 1 resulted in afluorescence increase at 530 nm and only gallium (III) ion was capableof selectively indicating phosphoproteins when bound to compound 1.Therefore, gallium (III) ion is the most preferred metal ion forphosphoprotein detection. This methodology was extrapolated to identifyother compounds wherein a different dye attached to the metal-chelatingmoiety.

(B) Differential Binding Affinity of Compound 1 for Phosphate Compounds.

Compound 1 complexed with gallium (III) ion has differential affinitiesfor various phosphate substrates in 75 mM NaOAc (pH 4.0) and 140 mMNaCl. Some of the phosphate-containing compounds studied were inorganicphosphate, phosphate attached to a protein, a peptide or an amino acid,pyrophosphate, ATP, and DNA. The affinities for these phosphate-basedsubstrates for the Compound 1/gallium (III) ion reagent were determinedto be ˜50 μM for inorganic phosphate and phosphate attached to aprotein, a peptide or an amino acid, ˜200 nM for pyrophosphate and ATP,and no binding was detected for DNA. Compare these values to theaffinity of compound 1 for gallium (III) ion of 2.5 μM. Most knownphosphate compounds should fall into one of these three categories withrespect to how it will bind to BAPTA gallium (III) ion; 1) singlephosphate group (i.e., inorganic phosphate or phosphate on a protein),2) multiple linked phosphate group (i.e., pyrophosphate or ATP), or 3)bridging phosphate group (i.e. nucleic acids).

(C) Compound 4 Displays Dual-Emission Wavelengths Upon SimultaneouslyBinding to Gallium (III) Ion and Phosphate.

Concentrations of 0.1-1.0 μM of compound 4 in a solution of 75 mM NaOAc(pH 4.0) and 140 mM NaCl display a single emission peak centered at 410nm (excitation 350 nm). Addition of 10 nM to 1 mM gallium chlorideresults in a decrease in the 410 nm emission and a concomitant increasein emission at 490 nm, with an isosbestic point of 475 nm. Thehalf-maximal response for this transition from the blue to greenemitting state occurs at approximately 1.8 μM gallium chloride.Therefore, 0.1 μM compound 4 with 1.7 μM gallium chloride in 75 mM NaOAc(pH 4.0) and 140 mM NaCl display both the 410 nm and 490 nm emissionpeaks. The addition of phosphate can alter the equilibrium between theemission peaks in favor of the longer wavelength 490 nm peak.

(D) Screening for Phosphate-Binding Compounds that Simultaneously BindGallium and Immobilized Phosphorylated Target Molecules.

A panel of test proteins was loaded on a denaturing SDS polyacrylamidegel, separated by electrophoresis, and the gels were fixed with 45%methanol, 5% acetic acid. Typically the test gels contained 500-600 ngeach of myosin, β-galactosidase, phosphorylase b, ovalbumin (2phosphates), carbonic anhydrase, soybean trypsin inhibitor, lysozyme,aprotinin, α₂-macroglobulin, phosphorylase b, glucose oxidase, bovineserum albumin, α₁ acid glycoprotein, carbonic anhydrase, avidin, andlysozyme. The gels also contained a 4-fold dilution series of α-casein(8 phosphates), 500 ng to 2 ng loaded. Thus the gels contained a rangeof proteins with different physicochemical properties, such as proteinswith hydrophobic binding pockets (e.g. BSA), glycosylated proteins (e.g.α₂-macroglobulin, glucose oxidase and avidin), acidic proteins (e.g.soybean trypsin inhibitor), basic proteins (e.g. lysozyme andaprotonin), and two different phosphoproteins (ovalbumin, α-casein). Thedilution series of α-casein yielded an estimate of phosphoproteinstaining sensitivity. A selection of phosphate-binding compoundscomprising different dye labels and different chelating moieties wasinitially screened in minimal binding buffers of pH 3.0 to 7.0, with avariety of metal ions, in the presence or absence of metal ion. Dye andmetal ion concentrations ranged from 0.1 to 10 μM, typically 0.3 to 3μM, and most frequently at 1.0 μM. Binding conditions that producedpreferential staining of phosphoproteins typically were at pH 3.0 to5.5, in the presence of certain trivalent metal ions. Under theseconditions, optimal preferential phosphoprotein staining was obtainedwith certain dye labels and the BAPTA chelating moiety with an equimolarconcentration of Ga³⁺. Further evaluation of the successful dyesrevealed that the pH optimum was 4.0, and that addition of salt (e.g.250-750 mM NaCl) improved staining specificity chiefly by decreasingintensity of staining of non-phosphoproteins. A broadened screen of dyeswas undertaken with 1 μM candidate dye, 1 μM of Ga³⁺ in 50 mM sodiumacetate, pH 4.0, 500 mM sodium chloride.

(E) Binding Solution Formulation

The binding solution comprises a phosphate-binding compound with a metalion in molar ratios of 1:2 to 2:1 and a buffer at about pH 3.0 to 6.0.Typically, the binding solution comprises a pH 3.0 to 5.5 buffer (50 to100 mM), salt (e.g. 100 to 1000 mM NaCl, or 100 to 300 mM MgCl₂) andequimolar concentrations of Ga³⁺ and of a phosphate-binding compound(e.g Compound 2), typically 1 to 10 μM each for detection purposes.Concentrations of the metal ion and phosphate-binding compound aretypically at least 100 times higher concentration for isolation purposesthan is present in the binding solution for detection purposes (see,Example 13). An optimal binding solution for a gel stain was prepared asfollows: 500 μg of compound 2 was dissolved in 873 μl water for a 1 mMstock solution. Five g of GaCl₃ were dissolved in 28.4 mL water for a 1M solution, from which 1 mL was combined with 9 mL water to make a 0.1 Msolution, from which 10 μL was added to 990 μL water for a 1 mM stocksolution. One liter of a 1 M stock solution of sodium acetate, pH 4.0was prepared by dissolving 136 g of sodium acetate trihydrate in ca. 800ml water, adjusting pH to 4.0 by adding ca. 23.5 mL 12 M HCl andbringing volume to 1 liter. One liter of a 4 M stock solution of sodiumchloride was prepared by dissolving 233.8 NaCl in ca. 800 mL water andbringing the volume to 1 liter with water. The sodium acetate and sodiumchloride stock solutions were filtered through a 0.45 μM filter. For 100mL of binding solution, 5 ml of 1 M sodium acetate, pH 4.0, 12.5 ml of 4M sodium chloride, and 20 mL of 1,2 propanediol were combined with waterto a final volume of 100 mL, to which was added while stirring 100 μL of1 mM GaCl₃ and 100 μl of 1 mM Compound 2 to obtain a final bindingsolution of 1 μM Compound 2,1 μM Ga³⁺, 20% 1,2-propane diol, 500 mMNaCl, 50 mM sodium acetate, pH 4.0.

Example 2 Detection of Phosphoproteins in SDS-Polyacrylamide Gels

Phosphoproteins were separated by SDS-polyacrylamide gel electrophoresisutilizing a 4% T, 2.6% C stacking gel, pH 6.8 and 13% T, 2.6% Cseparating gel, and pH 8.8, according to standard procedures. % T is thetotal monomer concentration (acrylamide+crosslinker) expressed in gramsper 100 mL and % C is the percentage crosslinker (e.g.,N,N′-methylene-bis-acrylamide, N,N′-diacryloylpiperazine or othersuitable agent). The separating gels were 8 cm wide by 5 cm high and0.75 cm in thickness. After electrophoresis, the gels were fixed byimmersing them in 100 mL 45% methanol and 5% acetic acid for 90 minutes.The gels were washed twice in water for a total of 30 minutes. The gelswere then added to a binding solution of the invention (Example 1E) andincubated for 120 minutes at room temperature with gentle orbitalshaking, typically 50 rpm. The binding buffer contained 50 mM NaOAc (pH4.0), 250 mM sodium chloride, 20% v/v 1,2-propanediol, 1 μM galliumchloride. To prepare the binding solution, 120 μL of a 1 mM stocksolution of Compound 2 and 120 μL of a 1 mM stock solution of galliumchloride were added to 1080 μL water. This mixture was then added to 59mL of the binding buffer to yield the binding solution. Alternatively,the phosphate-binding compound and the gallium chloride can be addedseparately, directly to 60 mL of the binding diluent. Binding solutionsthat utilize other phosphate-binding compounds of the present inventioncan be prepared and similarly tested for gel staining. After incubationin binding solution, the gel was washed with 75 mL of 50 mM NaOAc (pH4.0) and subjected to two washes of 30 minutes each.

For Compound 2 and other dyes that can be excited at 532 nm, images wereacquired on a Fujifilm FLA 3000 laser scanner using 532 nm excitationand 580 nm bandpass emission filters. For fluorescent phosphate-bindingcompounds that absorb in the ultraviolet or at visible wavelengths below532 nm, excitation was performed using 300 nm and detection was viaRoche Lumi-Imager or Fujifilm FLA 3000 laser scanner using 473 nmexcitation and 580 nm bandpass emission. The data were displayed usingImage Gauge Analysis software. Images of phosphoproteins were displayedas dark bands. Proteins not containing phosphate were not labeled orwere very lightly stained relative to the phosphoproteins. When gelswere labeled as above but with gallium chloride omitted from the bindingsolution, phosphoproteins were not selectively stained, and could not bedistinguished from background or had very light nonspecific staining.Gels were washed overnight with 50 mM NaOAc (pH 4.0) and images wereacquired as above. The background and nonspecific staining was furtherreduced relative to phosphoprotein staining. Replacement of galliumchloride by other gallium salts gave comparable results with allindicators tested; however, replacement by other metals, including Fe³⁺and Al³⁺ typically gave inferior results in staining of phosphoproteins.

Fixation of the gels in methanol/acetic acid can be done overnight orthe gels can be left in fixative for several days. Other salts can beused instead of sodium chloride, including magnesium chloride, magnesiumsulfate, and ammonium sulfate. Sodium chloride concentration ispreferably between 100 mM to 1000 mM. If salt is not included in thebinding solution, nonspecific staining of nonphosphoproteins isincreased. Nonspecific staining is reduced to low levels by extensivewashing with ˜50 mM NaOAc (pH 4.0). Buffers other than NaOAc may beused, including formate and 2-(N-morpholino)ethanesulfonic acid. If1,2-propanediol is omitted, the background staining of the gel isincreased but phosphoproteins are still selectively stained. The mosteffective pH ranges of the acidic buffers are in the range of 3.0 to6.0.

Example 3 Serial Dichromatic Detection of Phosphoproteins and TotalProtein in SDS Polyacrylamide Gels

After detection of the phosphoproteins as in Example 2, the gel wasincubated overnight with 60 ml SYPRO® Ruby protein gel stain (MolecularProbes, Eugene, Oreg.) with gentle orbital shaking, typically 50 rpm.The gel was then incubated in 7% acetic acid, 10% methanol for 30minutes, also at 50 rpm. The orange signal from the phosphorylated andnon-phosphorylated proteins was collected with a standard CCDcamera-based imaging system with 300 nm UV light excitation and a 600 nmbandpass filter.

Example 4 Detection of Phosphopeptides in a Polyacrylamide Gel

Peptides generated by a trypsin digestion of bovine milk β-casein wereseparated by electrophoresis in a Novex® Tricine gel (16%polyacrylamide, Invitrogen™ life technologies). After electrophoresisthe gel was fixed for 1 hour in 100 mL 40% methanol, 10% acetic acid,and then fixed for 1 hour in 100 mL of 40% methanol, 0.82 M NaOAc, 0.5%glutaraldehyde. The gel was washed with three changes of water, and thenincubated for 100 minutes in 30 mL staining solution containing 50 mMNaOAc, pH 4.0, 500 mM sodium chloride, 1 μM compound 2, 1 μM galliumchloride. The gel was then washed with three changes of 50 mM NaOAc in75 minutes. Images were acquired on a Fujifilm FLA 3000 laser basedscanner with 532 nm excitation and 580 nm bandpass emission filter anddata displayed using the Image Gauge Analysis software. The two knownphosphopeptides that result from a trypsin digest of β-casein werevisible as prominent bands on the gel. The gel was then stained with 60mL SYPRO® Ruby protein gel stain by incubating the gel overnight in thestain, and then incubating the gel in 7% acetic acid, 10% methanol for30 minutes.

Example 5 Detection of Phosphoproteins in Isoelectric Focusing Gels

Isoelectric focusing (IEF) can be performed utilizing a variety ofpre-cast and laboratory prepared gels that employ different chemistriesto generate a pH gradient. In this instance, Ampholine PAG plates wererun horizontally for 1500 volt-hours using a Multiphor IIelectrophoresis unit (Amersham-Pharmacia Biotech, Uppsala, Sweden) perthe manufacturer's instructions. The gels were fixed in 100 mL of 45%methanol, 5% acetic acid overnight. The gels were then washed withseveral changes of equal volumes of water, and incubated for 130 minutesin 50 mL of staining solution containing 50 mM2-(N-morpholino)ethanesulfonic acid (pH 3.0), 1000 mM NaCl, 1 μMcompound 2, and 1 μM gallium chloride. The gels were washed with 50 mLof 50 mM 2-(N-morpholino)ethanesulfonic acid (pH 3.0), 1 M NaCl twicefor 30 minutes per wash, and then in 50 mM2-(N-morpholino)ethanesulfonic acid (pH 3.0). Images were acquired asdescribed in Example 2.

Example 6 Detection of Phosphoproteins in Two-Dimensional Gels

A human MRC-5 lung fibroblast cell lysate protein mixture (150 μg) wasdiluted into urea buffer (7 M urea, 2 M thiourea, 2% CHAPS, 1%Zwittergent 3-10, 0.8% carrier ampholytes (3-10), 65 mM DTT) and appliedon a first dimension IPG strip (3-10 nonlinear, 18 cm). After overnightrehydration, the strips were covered with mineral oil and the proteinswere focused for 75,000 volts total. IPG strips were then laid on top of1 mm thick, 20 cm×20 cm, 12.5% T, 2.6% C polyacrylamide gels containing375 mM Tris base, pH 8.8 and SDS-polyacrylamide gel electrophoresis wasperformed according to standard procedures, except that the cathodeelectrode buffer was 50 mM Tris, 384 mM glycine, 4% SDS, pH 8.8 whilethe anode electrode buffer was 25 mM Tris, 192 mM glycine, 2% SDS, pH8.8. After the second dimension electrophoresis, gels were fixed in 750mL 45% methanol, 5% acetic acid for 20 hours. Gels were washed twice, 75minutes per wash, with water and then put in 500 ml staining solution.The staining solution contained 50 mM NaOAc, pH 4.0, 250 mM sodiumchloride, 20% v/v 1,2-propanediol, 1 μM compound 2, 1 μM galliumchloride. 500 μL of compound 2, in stock solution at 1 mM and 500 μL ofgallium chloride, in stock solution at 1 mM were added to 9 mL water.This mixture was then added to 490 mL of the staining buffer. The gelwas incubated for 8 hours in the binding solution; the solution wasdecanted and the gels were washed with 3 changes of 800 mL 50 mM NaOAc,pH 4.0, 30 to 40 minutes per wash, and then washed overnight in 1 liter50 mM NaOAc, pH 4.0. Images were acquired on a Fujifilm FLA 3000 laserscanner with 532 nm excitation and 580 nm bandpass emission filter anddata displayed using Image Gauge Analysis software. Images ofphosphoproteins were displayed as dark spots. Proteins not containingphosphate were not stained or were very lightly stained relative to thephosphoproteins. When gels were stained as above but with GaCl₃ omittedfrom the staining solution phosphoproteins were not selectively stained,and could not be distinguished from background or light nonspecificstaining. In addition, staining of phosphoproteins resulted in a trailof spots that correlated with different percentage of phosphorylation ofthe same protein, i.e., the protein had the same molecular weight butthe charge was different due to the addition or removal of a phosphategroup. Thus, 2-D gel analysis is a useful tool for identifyphosphoproteins using methods of the present invention and allows foridentification of changes in phosphorylation of a single protein.

Example 7 Serial Dichromatic Detection of Phosphoproteins and TotalProtein in 2-D Gels

Electrophoresis and phosphoprotein detection was performed as in Example6. After detection of the phosphoproteins, the gel was stained with 500mL SYPRO® Ruby protein gel stain by incubating the gel overnight in thestain, and then washing the gel in 7% acetic acid, 10% methanol for twochanges, at 30 minutes each wash. Images were acquired as described inExample 2. Alternatively, the orange signal from the phosphorylated andnonphosphorylated proteins is collected with a standard CCD camera-basedimaging system with 300 nm UV light excitation and a 600 nm bandpassfilter.

Example 8 Detection of Phosphoproteins Electroblotted to PVDF orNitrocellulose Membranes

Proteins of interest were separated by SDS-polyacrylamideelectrophoresis and transferred to PVDF membrane using standardprocedures, and the membrane was allowed to air dry. The PVDF membranewas quickly dipped in 100% methanol, washed with a solution of 40%methanol, 5% acetic acid for 15 minutes, and with two changes of waterfor 10 minutes each. The blot was then added to a binding solution andincubated for 80 minutes at room temperature with gentle orbitalshaking. The binding solution contained 50 mM NaOAc, pH 4.0, 500 mMsodium chloride, 1 μM Compound 1 or Compound 4, and 1 μM galliumchloride. Typically, 60 μL of the phosphate-binding compound, in stocksolution at 1 mM and 60 μL of gallium chloride, in stock solution at 1mM were added to 540 μL water. This mixture was then added to 29.5 mL ofthe staining buffer. Alternatively the phosphate-binding compounds andthe gallium chloride may be added separately, directly to 30 mL of thestaining diluent. After incubation in staining solution, the gel waswashed with 50 mL of 50 mM NaOAc; pH 4.0, 2 washes of 30 to 50 minuteseach.

Images were acquired with a standard CCD camera imaging system (BioRadFluorS Max) with a reflective 300 nm UV light source, and a 465 nmbandpass emission filter for Compound 4. Proteins not containingphosphate were not labeled or were very lightly stained relative to thephosphoproteins. When the blot was stained as above but with GaCl₃omitted from the staining solution, phosphoproteins were not selectivelystained, and could not be distinguished from background or lightnonspecific staining. For imaging with Compound 1, the wet blots wereplaced face down in the Fujifilm FLA 3000 laser scanner with a 473 nmexcitation laser and 520 nm bandpass emission filter, and data displayedusing the Image Gauge Analysis software.

Example 9 Dichromatic Detection of Phosphoproteins and Total ProteinElectroblotted to PVDF Membrane

Serial dichromatic detection of phosphoprotein and total protein on PVDFmembrane was accomplished by post-staining the blot labeled and imagedto detect phosphoprotein as in Example 8 (above) with SYPRO® Rubyprotein blot stain to detect total protein. The blot was floated facedown on a solution of 10% methanol, 7% acetic acid for 15 minutesfollowed by face staining with SYPRO® Ruby dye for 15 minutes. The blotwas washed face down on water, 3 changes in 10 minutes. The membrane wasallowed to air dry. The fluorescent signal from total proteins wasacquired with a standard CCD camera imaging system (BioRad FluorS Max)with a reflective 300 nm UV light source and a 610 nm longpass filter.

Dichromatic staining was achieved by image acquisition with a standardCCD camera imaging system (BioRad FluorS Max) with a reflective 300 nmUV light source and a 465 nm bandpass emission filter as in Example 8.The signal from the phosphoprotein stained with Compound 4/Ga (III)could be distinguished from the signal from total protein stained withSYPRO® Ruby, not detected with the 465 nm bandpass filter.

For Compound 1, SYPRO® Ruby staining and image acquisition as abovereveals fluorescent signal from total protein, revealing thephosphoproteins as a subset when the SYPRO® Ruby image is compared tothe fluorescent image obtained as in Example 6, above.

Example 10 Detection of Phosphatase Activity

Phosphoproteins and non-phosphorylated proteins were incubated withcommercially available calf intestinal alkaline phosphatase at 37° C.for 30 minutes under standard conditions. Control digests were doneunder the same conditions with no enzyme. Suitable test proteins includebovine α-casein, ovalbumin, and pepsin as phosphoproteins; and bovineserum albumin, chicken egg white lysozyme, and soybean trypsin inhibitoras non-phosphorylated proteins. Electrophoresis was performed as perExample 2, with control (undigested) and phosphatase-treated samplesloaded pairwise, 1250 ng protein per lane. Phosphoprotein detection wasperformed as per Example 2 above, with images taken 90 minutes afterlabeling and again after overnight washing. An additional gel waslabeled as per Example 2 but with no gallium chloride in the bindingsolution. For the gel labeled with the full binding solution,comparisons of the control, undigested sample proteins showed that thephosphoproteins appeared as dark bands according to the software displayand the nonphosphoproteins were not labeled or were only very lightlystained. For the gel labeled with the formulation lacking galliumchloride, phosphoproteins showed the same degree of no labeling or onlyvery light staining as the nonphosphoproteins, and this level of signalwas the same as the nonphosphoproteins in the gels labeled with the fullformulation including gallium chloride. Comparison of the pairwisephosphoproteins in the fully labeled gel showed that the signal from thealkaline phosphatase-treated sample was significantly less than thesignal from the undigested control. The very light signal from thenonphosphoproteins, if detectable, was virtually the same for thecontrol and enzyme-treated samples.

After detection of the phosphoproteins, the gel was stained for totalprotein with SYPRO® Ruby protein gel stain as per Example 2 and imagesof SYPRO® Ruby staining were acquired as per Examples 3 and 7. Thesignal for total protein staining was similar for the pairwise controland digested samples for both gels, indicating that the reduced signalfrom alkaline phosphatase-treated phosphoprotein samples was not due toprotein degradation.

Example 11 Detecting Kinase Activity

Bovine muscle myosin light chain was incubated with commerciallyavailable cloned calmodulin-dependent protein kinase II (New EnglandBioLabs) according to the manufacturer's instructions, with 100 mMadenosine triphosphate (ATP) and the supplied buffer components. Aparallel, control incubation was done with no enzyme. A sample of eachreaction mixture was loaded in adjacent lanes and analyzed byelectrophoresis as in Example 2. The gels were fixed in 100 mL of 45%methanol, 5% acetic acid for 60 minutes. The gels were then washed withseveral changes of water. One gel was incubated for 110 minutes in 30 mLof binding solution containing 50 mM 2-(N-morpholino)ethanesulfonicacid, pH 3.0, 1000 mM NaCl, 1 μM compound 2, 1 μM gallium chloride. Theother gel was incubated in an identical solution, minus galliumchloride. The gels were washed with 50 mL 50 mM2-(N-morpholino)ethanesulfonic acid, pH 3.0, 1000 mM NaCl twice for 30minutes per wash, and then in 50 mM 50 mM 2-(N-morpholino)ethanesulfonicacid, pH 3.0. Image acquisition for phosphoprotein detection was done asin Example 2 and serial dichromatic detection of phosphoproteins andtotal protein was done as in Example 3.

Staining for total protein revealed identical profiles of 3 major bandsin both lanes. Staining for phosphoprotein revealed one major band inboth lanes, with the signal from the band in the lane corresponding tothe reaction containing the enzyme 3.4-fold greater than the no-enzymecontrol.

Example 12 TRAIL/Apo2L Detection

To determine the cell signaling factors involved in TRAIL/Apo2L mediatedapoptosis, a proteomics approach involving 2-D gel electrophoresis andmass spectrometry is used. This approach involves comparing 2-D gels ofcolon cancer cells (Colo205) treated and not treated with a solublefragment of TRAIL/Apo2L (amino acids 114-281) for various lengths oftime ranging from several seconds to several hours. To assist incomparison of 2-D gels, compound 7 bound to gallium ions is used inconjunction with the SYPRO® Ruby total protein stain. Since cellsignaling often involves protein phosphorylation, the use of compound 7highlights spots likely to be involved in death receptor signaling orapoptotic signaling. Protein spots that are significantly differentbetween the TRAIL/Apo2L treated and untreated Colo205 cells areidentified by subsequent mass spectrometry analysis.

Example 13 Precipitation of Phosphopeptide

Mixtures of two non-phosphorylated peptides (Angiotensin I and II) andtwo phosphorylated peptides (pT/pY and RII) were combined (5 μL each) ina final volume of 100 μL containing 100 mM NaOAc, pH 4.0, 0.2 mM GaCl₃and 0.1 mM compound 9. The mixtures were vigorously vortexed for 1 hourat room temperature and then centrifuged in a microfuge at full speedfor 5 minutes. The supernatants were removed and stored. The pelletswere resuspended by triturating with a micropipet tip in 100 μL washbuffer (100 mM NaOAc, pH 4.0, 0.2 mM GaCl₃). The samples were againcentrifuged for 5 minutes and the supernatant wash components were savedfor analysis. The pellets were dissolved in 100 μL 50% acetonitrile,0.1% TFA for further analysis by HPLC or MALDI mass spectrometry.Pellets can also be dissolved in various different basic solutions ofchoice.

If phosphate-binding compound removal is required after precipitation,extraction with chloroform can be used. Also, any biotinylatedphosphate-binding compound, such as compound 9, can be used in theprecipitation procedure. After separation of the pellet, phosphopeptidesfrom the phosphate-binding compound/gallium complex using organic orbase treatment, the phosphate-binding compound can be removed using animmobilized streptavidin support (e.g., streptavidin-agarose orstreptavidin magnetic beads.)

Example 14 Detection of a Phosphopeptide in Solution by FluorescencePolarization using Compound 2-Ga³⁺

To demonstrate complexation of Compound 2-Ga³⁺ to phosphoproteins andphosphopeptides, fluorescence polarization of free dye was examined andcompared to the complex (compound 2-Ga³⁺) in the presence of aphosphorylated and non-phosphorylated protein and peptide.

First, an assay was conducted with a (1) phosphoprotein (ovalbumin) anda (2) control non-phosphorylated protein (lysozyme). A modified bindingsolution containing 1.0 μM Compound 2 was incubated in 50 mM2-(N-morpholino)ethanesulfonic acid (pH 3.0 to 3.5), 500 mM NaCl at roomtemperature in parallel to solutions containing, in addition, (a) 1 μMgallium chloride, (b) 100 μM lysozyme plus 1 μM gallium chloride, or (c)and 100 μM ovalbumin plus 1 μM gallium chloride. The fluorescencepolarization of the resulting solutions was then measured in afluorescence spectrophotometer with excitation at 530 nm and emission at545 to 700 nm. The integrated polarized emission spectra yieldedanisotropy “r values” of: r=0.10+/−0.003+/−0.02 (Compound 2 plusgallium); r=0.10+/−0.002 (Compound 2 plus lysozyme non-phosphorylatedcontrol); r=0.34+/−0.002 (Compound 2 plus gallium plus ovalbumin),indicating phosphorylation-dependent binding of the Compound 2 to thisphosphoprotein (see FIG. 10A)

Second, an assay was conducted with a (1) phosphopeptide, (2)non-phosphorylated peptide and (3) control with no peptide.Phosphorylated and non-phosphorylated delta sleeping inducing peptideDSIP (Typ-Ala-Gly-Gly-Asp-Ala-Ser (PO₃)-Gly-Glu) were purchased fromSynPep Corporation (Dublin, Calif.) Ovalbumin (Cat. A-7641) and lysozyme(Cat. L-7651) were purchased from Sigma Chemical Company (St. Louis,Mo.). For the assay 100 μM of each peptide and a peptide-free control inbinding solution (50 mM NaOAc pH 4.0, 500 mM NaCl, 1 μM Compound 2 and 1μM GaCl₃) were incubated for 30 minutes at room temperature. Thefluorescence polarization and anisotropy measurements were made using anAminco-Bowman Series-2 Spectrometer (Spectronic Instruments, Inc.,Rochester, N.Y.) using wavelength settings excitation 555±4 nm andemission wavelength setting 580 nm±4 nm. Alternatively, fluorescence wasmeasured with the Wallac 1420 Multilabel Counter (PerkinElmer LifeSciences) using wavelength settings excitation 535±17.5 nm and emissionwavelength setting 590 nm±17.5 nm. The binding solution alone andbinding solution in the presence of non-phosphorylated peptidedemonstrates very similar fluorescence polarization and anisotropies.However, in the presence of the phosphopeptide there is a significantincrease in the fluorescence polarization and the anisotropy values.This result demonstrates specific binding of the phosphopeptide to theCompound 2-Ga³⁺ complex in solution but not the non-phosphorylatedpeptide. See FIG. 10B.

This assay also provides a method for screening compounds that will bindtrivalent gallium ions and label phosphorylated peptides and forsolution based kinase assays.

Example 15 Isolation and Characterization of Phosphopeptides fromComplex Protein Digests with a Matrix-Immobilized Phosphate-bindingcompound

A phosphate-binding compound-agarose column (compound 13 or 14)(typically 200 μL of medium) was charged with 0.1 M GaCl₃ and washedwith de-ionized H₂O until the pH of the flow-through material approached7.0. The column was then equilibrated with 5 column volumes of bindingbuffer (100 mM NaOAc buffer (pH 3.0)). The phosphopeptide mixture wasvacuum dried in the SpeedVac (Savant) or similar instrument anddissolved in binding buffer. If the final pH of the peptide mixture isnot 3.0, then it can be adjusted with 1-10 M acetic acid as appropriate.The protein digest (1-5 mg/mL) was applied in 1 column volume or less(but no less than half the column volume) and followed with 2 columnvolumes of binding buffer. Flow-through (FT) fractions were combined andstored for further analysis. The column was washed with 2 column volumesof 100 mM NaOAc (pH 7.0), 500 mM NaCl, 10% acetonitrile followed by 1column volume of NaOAc (pH 7.0). The FT fractions were combined andstored for further analysis. Bound peptides were eluted with 3 separatecolumn volumes of saturated Ba(OH)₂ that are collected in a single tube.The pH of the resulting elution fraction was greater than pH 11.0, andwhen it was not, it was immediately adjusted with saturated Ba(OH)₂. Theelution fraction was incubated for 90 minutes at 30° C. Afterincubation, the sample was divided into 2 portions, one of which wasneutralized to pH 5.0-7.0 with glacial acetic acid and stored frozen.One-half volume of de-ionized water is added to the other tube followedby the addition of a concentrated nucleophilic thiol or amine(methylamine, cystamine or β-mercaptoethylamine) to achieve a finalconcentration of 0.1-0.5 M in a volume not exceeding ⅙ of the startingsample/H₂O volume. The reaction mix was incubated for an additional 60minutes at 30° C., then neutralized to pH 5.0-7.0 with glacial aceticacid. For MALDI-TOF mass spectrometry analysis, peptides were purifiedfrom samples using C18 ZipTips (Millipore) using standard protocols,vacuum dried in a SpeedVac dryer and dissolved in 50% acetonitrile and0.1% TFA. An equal volume of 10 mg/mL MALDI matrix(α-cyano-5-hydroxycinnamic acid) in the same solvent was added. Thesolution was mixed thoroughly and 1 μL was spotted onto the MALDItarget.

Differential mass weight analyses of both peptide fractions resulted inthe determination of the number of phosphorylation sites on thepeptides, as well as the nature of the phosphoamino acids. Under theconditions used, only phosphoserine residues undergo elimination andnucleophilic addition (loss of phosphoric acid −98 amu, +mass weight ofnucleophilic addition reagent). Phosphothreonine residues undergoelimination only (loss of phosphoric acid only, −98 amu) andphosphotyrosine residues remain unchanged, as phosphotyrosine is stablein strong base.

Example 16 Quantitating the Number of Phosphates on Ovalbumin

Solutions of 1 μM and 4 μM ovalbumin were incubated in 75 mM NaOAc (pH4.0), 140 mM NaCl, 0.1 μM Compound 4, and 1.7 μM gallium chloride atroom temperature for 5-10 minutes. The fluorescence intensity of theresulting solution was then measured at 410 nm in a fluorescencespectrophotometer and compared to a standard phosphate calibration curveto determine the number of phosphates on ovalbumin. The standardphosphate calibration curve was produced by equilibrating knownconcentrations (1, 2, 4, 6, 8, and 10 μM) of a 19 amino acidphosphoserine-containing peptide in 75 mM NaOAc (pH 4.0), 140 mM NaCl,0.1 μM compound 4, and 1.7 μM gallium chloride and measuring thefluorescence intensity at 410 nm. Next the fluorescence intensity wasgraphed versus the known concentration of phosphopeptide. Thefluorescence intensity from the solution containing ovalbumin wascompared to the standard curve to reveal ˜2 μM and ˜8 μM phosphate.Finally, accounting for the protein's concentration resulted in thedetermination of two phosphate groups per molecule of ovalbumin.

Example 17 Phospholipid Detection

To test the detection of phospholipids with the present invention,different phospholipids were spotted onto a nitrocellulose membrane. Thephospholipids were obtained from Echelon Research Labs in a formatcalled a PIP Array™, which contains 8 different phosphoinositides(Ptdlns) at 7 different concentrations. PIP Arrays™ were used fordetermining the sensitivity limits of the invention for detectingphospholipids.

PIP Array™

-   -   1. Ptdlns 100 50 25 12.5 6.3 3.2 1.6 pmol    -   2. Ptdlns (3) P    -   3. Ptdlns (4) P    -   4. Ptdlns (5) P    -   5. Ptdlns (3,5) P2    -   6. Ptdlns (4,5) P2    -   7. Ptdlns (3,4) P2    -   8. Ptdlns (3,4,5,) P3

One PIP Array™ was washed in 50 mM NaOAc (pH 4.0) for 15 min. After thewash, the PIP Array™ was incubated in 50 mM NaOAc (pH 4.0), 20%1,2-propanediol, 500 mM NaCl, 1 μM compound 1, and 1 μM GaCl₃ for 1hour, by incubating the array at 100-150 RPM on an orbital shaker. Afterincubating the PIP Array™ the array was washed 3 times in 50 mM NaOAc(pH 4.0) for 15 minutes each at 100-150 RPM on an orbital shaker toremove unbound dye and reduce the background fluorescence. An image ofthe PIP Array™ was generated using a laser based scanner (Fuji FLA 3000)with an excitation wavelength of 473 nm and an emission filter of 520nm. Of the eight phosphoinositides, four gave a strong positive signal.These included phosphatidic acid, phosphoinositide (4,5) P₂,phosphoinositide (3,4) P₂ and phosphoinositide (3,4,5) P₃. The strongestsignal was obtained with phosphoinositide (3,4) P₂ followed byphosphoinositide (4,5) P₂ and then phosphoinositide (3,4,5) P₃.

Example 18 Phosphoprotein Detection on Microarrays

Four specific, purified proteins including β-casein, ovalbumin, pepsinand bovine serum albumin were arrayed from a source plate (384 wellplate) at a concentration of 0.975 μg/mL-0.5 mg/mL in water, ontoHydroGel coated slides (Perkin Elmer), using the BioChip Arrayer™(Packard Instrument Co., Meriden, Conn.). The BioChip Arrayer™ utilizesa PiezoTip™ Dispenser consisting of 4 glass capillaries. Proteins weredispensed from the PiezoTip™ by droplets 333 pL in volume to createarray spots 175 microns in diameter with a 500 micron horizontal andvertical pitch (pitch=center to center spacing of spots). Proteins werearrayed in duplicate in four rows, with 10 dilution points, resulting inan array of 160 spots. The resulting concentration range of the arraywas 166.5 pg/spot -0.325 pg/spot. For detection of phosphoproteins,slides were incubated for 1 hour on a rotator in 1 μM of Compound 2, inbuffer containing 0.5 M NaCl, 20% 1,2-propanediol, 1 μM GaCl₃, and 0.05M NaOAc, pH 4.0. Slides were then washed for 1 hour on a rotator in 0.05M NaOAc, pH 4.0, containing 10% methanol followed by a 15 minute waterwash. Slides were then spun briefly in a microarray high-speedcentrifuge affixed with a rotor with a slide holder (Telechem) at ˜6000rpm to remove excess liquid. After the slides were dry, the arrays wereimaged using the ScanArray® 5000 XL Microarray Analysis System (PackardInstrument Co., Meriden, Conn.) using the 543.5 nm laser and either 570nm or 592 nm emission filter. Phosphate content per protein wasdetermined to be as follows: β-casein, five phosphates; ovalbumin, twophosphates; pepsin, one phosphate; and BSA, no phosphates.

Example 19 Phosphopeptide Detection on Microarrays

Two peptides, Kemptide and pDSIP, were arrayed on to HydroGel coatedslides (Perkin Elmer) from a source plate (384-well) with aconcentration of 0.03125 to 2 mg/mL peptide in water. The amino acidsequence of Kemptide is Leu-Arg-Arg-Ala-Ser-Leu-Gly (MW 771.9). Theamino acid sequence of pDSIP isTrp-Ala-Gly-Gly-Asp-Ala-Ser(PO₃H)-Gly-Glu (MW 929.5). Arrays werespotted using a manual glass slide arrayer (V & P Scientific, San Diego,Calif.) fixed with 4 rows of 8 pins (32 total), ˜500 micron diameterspot size, 1.125 micron horizontal pitch and 750 micron vertical pitch(pitch=center to center spacing of spots). The hand arrayer collected 6nL of peptide from the source plate and transferred ˜6 nL to the arraysurface by direct contact. The resultant peptide concentration was 0.18to 12 ng/spot. Peptides were arrayed in replicates of 6, resulting in anarray of 84 spots. For specific detection of pDSIP, the phosphopeptide,slides were incubated for 1 hour on a rotator in 1 μM dye of compound 2in buffer containing 0.5 M NaCl, 20% 1,2-propanediol, 1 μM GaCl₃, and0.05 M NaOAc, pH 4.0. Slides were then washed for 1 hour on a rotator in0.05 M NaOAc, pH 4.0, containing 10% methanol followed by a 15-minutewater wash. Slides were then spun briefly in a microarray high-speedcentrifuge affixed with a rotor with a slide holder (Telechem) at ˜6000rpm to remove excess liquid. After the slides were dry, the arrays wereimaged using the ScanArray® 5000 XL Microarray Analysis System (PackardInstrument Co., Meriden, Conn.) using the 543.5 nm laser and either 570nm or 592 nm emission filter.

Example 20 Detection of Immobilized Kinase Substrates in MicroarrayFormat; Selective Detection of Glycogen Synthase 1-10

Two specific peptides, Abl peptide and glycogen synthase 1-10, werearrayed from a source plate (384-well plate) concentration of 0.03-2mg/mL in water, onto HydroGel coated slides (Perkin Elmer). Abl peptide(New England Biolabs) is a substrate for Abl tyrosine kinase and itsamino acid sequence is E-A-I-Y-A-A-P-F-A-K-K-K (MW 1336). Glycogensynthase 1-10 (Calbiochem) is a substrate forCalcium-Calmodulin-Dependent protein Kinase II and its amino acidsequence is P-L-S-R-T-L-S-V-S-S (MW 1045.2). Arrays were spotted using amanual glass slide arrayer (V&P Scientific, San Diego, Calif.) fixedwith 4 rows of 8 pins (32 total), ˜500 micron diameter spot size, 1.125micron horizontal pitch and 750 micron vertical pitch(pitch=center-to-center spacing of spots). The handarrayer collected 6nL of peptide from the source plate and transferred ˜6 nL to thehydrogel coated slide by direct contact. The resultant peptideconcentration is 0.18 to 12 ng/spot. Peptides were arrayed in replicatesof 6, resulting in array of 96 spots (12 spots, of which were 0ng/spot). Slides were left overnight after arraying in a humiditychamber. Slides were then blocked for 1 hour in 100 mM HEPES, 1% BSAwhile rotating (Barnstead/Thermolyne Labquake rotisserie). Afterblocking, the slides were spun briefly in a small microarray high-speed(max ˜6000 rpm) centrifuge affixed with a rotor with a slide holder(Telechem) to remove excess liquid. Next, kinase reactions wereperformed by attaching a Grace Biolabs Hybriwell™ hybridization sealingsystem (40×22×0.25 mm) to the hydrogel coated slide to enclose the areacontaining the hydrogel polyacrylamide pad. The reaction was carried outin an 80 μL reaction volume containing 20,000 U/mL or 1600 units enzyme(Calmodulin-Dependent protein Kinase II, NEB) using buffer, CaCl₂,calmodulin, and ATP supplied with the enzyme. 1× CamKII buffer included50 mM Tris-HCl, 10 mM MgCl₂, 2 mM dithiothreitol, 0.1 mM Na₂EDTA, pH7.5. CaCl₂, calmodulin and ATP working concentrations were 2 mM, 1.2 μMand 0.10 mM. The reaction solution with enzyme was pipetted into theHybriwell™ through 1 of 2 ports on the seal cover. Ports were thensealed with seal-tabs, placed in a CMT-hybridization chamber (VWRScientific) and incubated on a nutator (Clay Adams) in a 37° C.incubator. The kinase reaction was carried out for 3 hours. Afterincubation, the slides were removed from the hybridization chamber andwashed 2 times for 5 minutes in 10% SDS followed by 5-7 times for 5minutes in water while rotating. Slides were then transferredimmediately to binding solution comprising 1 μM of compound 2 in 50 mMNaOAc, pH 4.0; 500 mM NaCl; 20% 1,2-propanediol; and 1 μM GaCl₃ for 45minutes while rotating. Slides were then washed 3 times for 15 minuteseach time in 50 mM NaOAc, pH 4.0, 10% methanol followed by a 15-minutewater wash. Slides were then dried and imaged using the Scan Array® 5000XL Microarray Analysis System (Packard Instrument Co., Meriden, Conn.)using the 543.5 nm laser and either 570 nm or 592 nm emission filters.Calmodulin-dependent kinase II specifically phosphorylates glycogensynthase 1-10 peptide. Using the 543.5 nm excitation and 570 nm emissionfilter, glycogen synthase peptide is the only fluorescently labeledpeptide on the array. Sensitivity of detection after kinase reaction isat least 0.375 ng or 0.35 pmol.

Example 21 Detection of Immobilized Kinase Substrates in MicroarrayFormat; Specific Detection of Abl Peptide Substrate

The following experiment was performed essentially as described inExample 20 with the following differences. Two specific peptides, Ablpeptide and Kemptide, were arrayed from a source plate (384-well plate)concentration of 0.03 to 2 mg/mL in water, onto hydrogel-coated slides(Perkin Elmer). Kemptide (New England Biolabs) is a substrate forcAMP-dependent Protein Kinase (PKA) catalytic subunit and its amino acidsequence is L-R-R-A-S-L-G (MW 771). Arrays were spotted as described inExample 20 and kinase reactions performed as described previously. Thereaction was carried out in a 80 μL reaction volume containing 3,750U/mL or 300 units enzyme (Abl Protein Tyrosine Kinase, NEB) using bufferand ATP supplied with the enzyme. 1× Abl buffer included 50 mM Tris-HCl,10 mM MgCl₂, 1 mM EGTA, 2 mM dithiothreitol, 0.01% Brij 35, pH 7.5.Labeling of slides and imaging were performed as previously described.Using the 543.5 nm excitation and 570 nm emission filter, Abl peptidesubstrate is the only fluorescently labeled peptide on the array.Sensitivity of detection after kinase reaction is at least 0.18 ng or0.14 pmol.

Example 22 Ratiometric Analysis of Phosphorylated Target molecules usinga Binding solution of the Present Invention and the Total Protein StainSYPRO® Ruby Gel Stain

SDS-polyacrylamide gels were loaded with serial dilutions ofphosphoproteins and non-phosphoproteins and gels stained as described inExample 2. The gels were illuminated and fluorescence signal quantified(intensity) for each protein concentration and each stain(phosphoprotein and total protein). The ratio of these fluorescenceintensities was then graphed, phosphoprotein and total protein, againstthe amount of protein loaded in the well (ng). The fluorescenceintensity for each stain was next plotted against the proteinconcentration, this graph allows for the calculation of a constantnumber for each of the two stains, the Y-intercept value. TheY-intercept value is then subtracted from the fluorescence intensityvalues and the resulting rations (phosphoprotein to total protein) areagain graphed against the protein concentration. This resulting graphproduces numbers wherein stained phosphoproteins have ratio values50-100 times greater than nonphosphorylated proteins, thus nonspecificstaining and low abundance phosphoproteins can be distinguished fromnon-phosphorylated proteins. See, FIG. 10.

Example 23 Incorporation of ATP-γ-S During Phosphorylation ofImmobilized Peptides on Microarrays and Subsequent Detection of thePhosphorothioate Group with a Binding Solution of the Present Invention

Three specific peptides including Abl peptide, Kemptide and GlycogenSynthase 1-10 were arrayed from a source plate (384-well plate)concentration of 0.03 μg/mL to 0.5 mg/mL in water, onto hydrogel-coatedslides (Perkin Elmer). Proteins were spotted using the BioChip Arrayer™(Packard Instrument Co., Meriden, Conn.) that utilizes a PiezoTip™Dispenser consisting of 4 glass capillaries. Proteins were dispensedfrom the PiezoTip™ by droplets 333 pL in volume to create array spots175 microns in diameter with a 500 micron horizontal and vertical pitch(pitch=center to center spacing of spots). Each peptide was arrayed inquadruplicate from a 2-fold dilution series consisting of 15 dilutionpoints, resulting in an array of 240 spots (including 60 water spots=0pg/spot protein). The resultant peptide concentration was 0.01 pg/spotto 166.5 pg/spot. Slides were left overnight after arraying in ahumidity chamber. Slides were then blocked for 1 hour in 100 mM HEPES,1% BSA, pH 7.5, while rotating (Barnstead/Thermolyne Labquakerotisserie). After blocking, the slides were spun briefly in a smallmicroarray high-speed (max ˜6000 rpm) centrifuge affixed with a rotorwith a slide holder (Telechem) to remove excess liquid. Next, kinasereactions were performed by attaching a Grace Biolabs Hybriwell™hybridization sealing system (40×22×0.25 mm) to the hydrogel-coatedslide to enclose the area containing the hydrogel acrylamide pad. Thereaction was carried out in a 80 μL reaction volume containing 3750 U/mLor 300 units enzyme (Abl Protein Tyrosine Kinase, NEB) using buffersupplied with the enzyme. 1× Abl buffer included 50 mM Tris-HCl, 10 mMMgCl₂, 1 mM EGTA, 2 mM dithiothreitol, 0.01% Brij 35, pH 7.5. ATP-γ-S(Sigma Chemical Company) was substituted in place of ATP at a workingconcentration of 0.10 mM. A control reaction was run simultaneously on asecond slide using ATP itself, supplied with the enzyme, at a workingconcentration of 0.10 mM. The reaction solution containing the enzymewas pipetted into the Hybriwell™ through 1 of 2 ports on the seal cover.Ports were then sealed with seal-tabs, placed in a CMT-hybridizationchamber (VWR Scientific) and incubated on a nutator (Clay Adams) in a37° C. incubator. The kinase reaction was carried out for 3 hours. Afterincubation, the slides were removed from the hybridization chamber andwashed 2 times for 5 minutes in 10% SDS followed by 7 times for 5minutes in water while rotating. Slides were transferred immediately to1 μM compound 2 in 50 mM NaOAc, pH 4.0, 500 mM NaCl, 20%1,2-propanediol, 1 μM GaCl₃ for 45 minutes while rotating. Slides werewashed 3 times for 15 minutes each time in 50 mM NaOAc, pH 4.0, 4%acetonitrile, followed by a 15 minute water wash. Slides were dried andimaged using the Scan Array® 5000 XL Microarray Analysis System (PackardInstrument Co., Meriden, Conn.) using the 543.5 nm laser and either 570nm or 592 nm emission filters. Abl Protein Tyrosine Kinasephosphorylates the tyrosine residue of the Abl peptide substrate withthe phosphorothioate (or phosphate as in control). Using the 543.5 nmexcitation and 570 nm emission filter, Abl peptide substrate is the onlyfluorescently labeled peptide on the array. Sensitivity of detectionafter kinase reaction was 2.6 pg of the peptide labeled using ATP-γ-S.By contrast, 0.325 pg of Abl peptide, in the control reaction using ATP,was labeled and detected with binding solution containing Compound 2.

Example 24 Isolation of Phosphopeptides Via Immobilized Streptavidin orPhosphate-Binding Compound on Membranes

Immunodyne membranes, purchased from Pall, were labeled by adding enough5-10 mg/mL streptavidin or BAPTA-amine to wet the membrane(approximately 15 μL per cm²). After air-drying for 15-30 minutes themembranes were washed 3 times for 5 minutes with 50% Acetonitrile/0.1%TFA, then twice for 5 minutes with water. Experiments were performedusing 6 mm diameter circles of membrane. Streptavidin-labeled membraneswere charged by soaking in 200 μL of 40 μM Compound 9/250 μM GaCl₃, andthe BAPTA labeled membranes were charged by soaking in 200 μL 250 uMGaCl₃. After washing once with 10% acetonitrile/400 mM NaCl, 100 mMNaOAc, pH 4, then twice with water, the membranes were soaked in 100 μLof peptide mixture containing 2 μg each of 2 non-phosphopeptides and 3phosphopeptides (approximately 6 nanomoles of total peptide) for 15 min.The membranes were washed once with 10% acetonitrile/400 mM NaCl/100 mMNaOAc, pH 4, then twice with 100 mM NaOAc, pH 4, for 5 min per wash.Finally the membranes were eluted with 100 μL of 50% acetonitrile/0.1%TFA. The eluates and peptide supernatants (peptide solution afterincubation of membranes) were prepared for MALDI analysis.

Example 25 Isolation of Phosphopeptides with Immobilized Streptavidin onFerrofluid Beads

A. Isolation of Phosphopeptides Using Streptavidin Ferrofluid MagneticParticles in Conjunction with BAPTA-Biotin Dyes.

Streptavidin ferrofluid magnetic particles were used in conjunction withBAPTA-biotin compounds to isolate phosphopeptides from a complexmixture. The magnetic particles were labeled with Compound 9 and twoversions of rhodamine-biotin dyes (Compound 15 and Compound 25) due tothe binding between biotin and streptavidin. After washing awayphosphate-binding compounds, the particles were charged with 0.1 MGaCl₃, then washed with 0.1 M NaOAc, pH 4.0. Fifty μL of the magneticparticle slurry (4 mg/mL) was added to the phosphopeptide mix in a totalvolume of 100 μL. The particles were incubated with gentle vortexing for20 minutes and the particles were isolated using a magnetic separator.The supernatants were removed and the beads were washed 2× with 100 μL100 mM NaOAc, pH 4.0. The phosphopeptides were eluted with 100 μL 50%acetonitrile, 0.1% TFA. The isolated peptides were analyzed by MALDI anddemonstrated that only phosphorylated peptides were isolated.

B. Quantitative Binding of Phosphopeptides Using Streptavidin FerrofluidParticles

Quantitative binding of phosphopeptides was accomplished using labeledferrofluid particles. Streptavidin ferrofluid particles were labeledwith Compound 9 until saturated. After washing away unbound dye, theparticles were charged with 0.1 M GaCl₃, then washed with 0.1 M NaOAc,pH 4.0. 100 μL of magnetic particles (4 mg/mL) was added with standardpeptide mix containing 550 picomoles of phosphopeptides. The mixture wasincubated for 20 min while gently vortexing. The particles were isolatedusing a magnetic separator and the resulting supernatant was removed.The beads were washed 2× with 100 μL 100 mM NaOAc, pH 4.0 and thephosphopeptides were eluted with 0.15 M ammonium hydroxide. Greater than95% of the phosphopeptides (550 pmoles) were isolated.

C. Ferrofluid Particle Isolation of Phosphopeptides Coupled with BaseElimination/Addition

Streptavidin ferrofluid (StFF) particles labeled with Compound 9 wereused for phosphopeptide isolation and coupled with baseelimination/addition. Phosphopeptides were isolated on StFF and thephosphopeptides eluted with 50 μL of 0.15 M Ba(OH)₂. Subsequently, 3 μLmethylamine was added to the eluate and peptides were incubated at 37°C. for 1 hour. The reaction was neutralized with glacial acetic acid topH 4.0 and the eluted peptides were desalted with ZipTips. The use ofstreptavidin-labeled ferrofluid beads allows for a strong interactionbetween avidin and biotin that facilitates isolation of largerphosphorylated target molecules.

Example 26 Precipitation of Phosphopeptides with DTPA Compounds

Phosphate-binding compounds comprising DTPA (Compounds 20, 21 and 22)were used in a precipitation reaction for the isolation ofphosphopeptides from complex solutions. Precipitation reactionscontained 100 μM Compound 20, 21 or 22, 200 μM GaCl₃, 100 mM NaOAc, pH4.0 and 5 μL of an 8-peptide mix (250 ng/μL each). The samples werevigorously vortexed for 20 minutes then centrifuged at 14,000 rpm for 5minutes. The supernatants were removed and stored and the pellets werewashed 2× in 100 mM NaOAc, pH 4.0 by resuspension with a pipet tip andre-centrifuging. The final pellets were dissolved in 100 μL of 50%acetonitrile, 0.1% TFA. Supernatants and pellet fractions were diluted1:100 in 50% acetonitrile, 0.1% TFA and then mixed 1:1 with MALDI matrixand then spotted onto a MALDI target. The MALDI analyses demonstratedthe selective isolation of phosphorylated peptides.

Example 27

Serial detection of total phosphopeptide content with a binding solutionof the present invention followed by specific detection ofphosphotyrosine residues in peptides using a phosphotyrosine specificmonoclonal antibody and (A) Alexa Fluor® 647 goat anti-mouse or (B)Zenon™ One Alexa Fluor® 647 mouse IgG labeling reagent, both inmicroarray format.

(A) Three pairs of peptides, the phosphorylated and thenon-phosphorylated forms of Kemptide, pp60 c-src and DSIP, were arrayedon to HydroGel coated slides (Perkin Elmer) from a source plate(384-well) with a concentration of 0.95 μg/ml -0.5 mg/ml peptide inwater, using the BioChip Arrayer™ (Perkin Elmer) as described in Example20. For specific detection of the phosphopeptides on the array, theslides were incubated for 45 minutes on a rotisserie in a bindingsolution comprising 1 μM Compound 2, in buffer containing 0.5 M NaCl,20% 1,2 propanediol, 1 μM GaCl₃, and 0.05 M sodium acetate, pH 4.0.Slides were then washed three times for 15 minutes on a rotisserie in0.05 M sodium acetate, pH 4.0, containing 4% acetonitrile followed by a15 minute water wash. Slides were then spun briefly in a microarrayhigh-speed centrifuge affixed with a rotor with a slide holder(Telechem) at ˜6000 rpm to remove excess liquid. After slides were dry,the arrays were imaged using the ScanArray® 5000 XL Microarray AnalysisSystem (Packard Instrument Co., Meriden, Conn.) using the 543.5 nm laserand 570 nm emission filter. The binding solution specifically labeled1.3-2.6 pg pDSIP, 2.6-5.2 pg pKemptide and 10.4-20.8 pg pp60 c-src (pY).Following phosphopeptide detection, the slides were immediately placedin blocking buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 0.2%Tween-20, 0.25% MOWIOL-488, 0.5% BSA and incubated while rotating for3-5 hours. Slides were then transferred to blocking buffer (describedabove) containing a 1:1000 dilution (final concentration of 1.5 μg/mL)of phosphotyrosine monoclonal antibody (supplied at 1.5 mg/mL;P-Tyr-100; Cell Signaling Tech.) and incubated overnight at 4° C. whilerotating. After overnight incubation with the primary antibody, theslides were washed three times for 10 minutes in blocking buffer andthen incubated for 45 minutes, while rotating, in blocking buffercontaining a 1:5000 dilution (final concentration of 0.4 μg/mL) of AlexaFluor® 647 goat anti-mouse (supplied at 2 mg/mL). Finally, slides werewashed two times for 10 minutes in blocking buffer followed by two 5minute washes in 50 mM Tris, pH 7.5, 150 mM NaCl and spun briefly in amicroarray high-speed centrifuge. After the slides were dry, the arrayswere imaged using the ScanArray® 5000 XL Microarray Analysis System(Packard Instrument Co., Meriden, Conn.) using two protocols with a543.5 nm laser/570 nm emission filter set and a 632.8 nm laser/670 nmemission filter set. Using the 543.5 nm excitation and 570 nm emissionfilter, there was no signal detected. Using the 632.8 nm excitation and670 nm emission filter, pp60 c-src (pY) was specifically detected to asensitivity of 5.2 pg.

(B) After overnight incubation with the primary antibody, the slideswere washed three times for 10 minutes in blocking buffer and thenincubated for 45 minutes, while rotating, in blocking buffer containinga 1:100 dilution (final concentration of 2 μg/mL) of Zenon™ One AlexaFluor® 647 mouse IgG labeling reagent (supplied at 200 μg/mL). Finally,slides were washed once for 5 minutes in blocking buffer, once for 5minutes in 50 mM Tris, pH 7.5, 150 mM NaCl and spun briefly in amicroarray high-speed centrifuge. After the slides were dry, the arrayswere imaged using the ScanArray® 5000 XL Microarray Analysis System(Packard Instrument Co., Meriden, Conn.) using two protocols with a543.5 nm laser/570 nm emission filter set and a 632.8 nm laser/670 nmemission filter set. Using the 543.5 nm excitation and 570 nm emissionfilter, there was no signal detected. Using the 632.8 nm excitation and670 nm emission filter, pp60 c-src (pY) was specifically detected to asensitivity of 0.65 pg.

Example 28 Size Exclusion Column (SEC) and Reverse Phase (RP)HPLCAnalysis of Phosphopeptides Using a Binding Solution of the PresentInvention

10-40 μL of sample containing 2-60 μM phosphopeptide (or a control of100 μm non-phosphopeptide), 20 μM Compound 2, 40 μM GaCl₃, 100 mM sodiumacetate pH 4 and 0-20% ethyl alcohol or isopropyl alcohol was injectedonto a size exclusion column (Superdex 30, 10×300 mm or Superdex Peptide3.2×300 mm). Mobile phase was 50 mM sodium acetate pH 4, 500 mM NaClplus 20% ethyl alcohol or isopropyl alcohol. The runtime was 45 min. UVand fluorescence signal was monitored at 214 nm and 555ex/580em,respectively.

SEC results demonstrated an enhanced fluorescent signal in the presenceof a binding solution of the present invention and a phosphopeptide (60μM) compared to a non-phosphopeptide.

The same sample was also analyzed by reverse phase HPLC with a Vydac238TP52, 2.1×250 mm C₁₈ column. Solvent A=100 mM sodium acetate pH 4,0-20% ethyl alcohol. Solvent B=100 mM sodium acetate pH 4, 20% ethylalcohol, 60% methanol. A gradient separation was performed, 0-55%solvent B over 30 min at 0.2 mL/min. UV (214 nm) and fluorescence (ex555/em 580) signals were monitored.

RP results demonstrated an enhanced fluorescent signal in the presenceof a binding solution of the present invention and a solution withoutgallium chloride. Samples were analyzed containing amono-phosphotyrosine, mono-phosphothreonine and mono-phosphoserinecontaining peptide, with comparable results.

Example 29 Detection of Phosphopeptides on Streptavidin-PolystyreneBeads Using a Binding Solution of the Present Invention

Streptavidin-polystyrene beads (4.0-4.9 μM) were charged with either oneof two biotinylated synthetic peptides, a phosphopeptide or anon-phosphopeptide. The phosphopeptide had a molecular weight of 1812g/mol and the amino acid sequence wasbiotinyl-ε-aminocaproyl-Glu-Pro-Gln-Tyr(PO₃H₂)-Glu-Glu-Ile-Pro-Ile-Tyr-Leu-OH.The non-phosphopeptide had a molecular weight of 2342.55 g/mol and theamino acid sequence wasbiotinyl-Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH₂.Beads were charged in 100 mM Tris, 100 mM NaCl, pH 7.5 and washedseveral times in the same buffer, following charging, before staining.Both sets of beads were then stained with 1 μM Compound 2 in buffercontaining 0.05 M sodium acetate, pH 4.0, 1 μM GaCl₃, 0.5 M NaCl, and20% 1,2 propanediol for 45 minutes. Following staining, beads werewashed in 0.05 M sodium acetate, pH 4.0, 4% acetonitrile and mixed indifferent ratios of phosphopeptide-charged beads with non-phosphopeptidecharged beads. All steps were performed with rotation and rigoroussonication and vortexing. The mixed bead populations were then imagedusing a Nikon Eclipse 800 Epi-Fluorescent Microscope using OmegaOptical, Inc. filter set XF102-2 (Exciter: 560AF55; Dichroic: 595DRLP;Emitter: 645AF75). The fluorescent signal of the phosphopeptide chargedbeads was found to be 6-fold higher, on average, than thenon-phosphopeptide charged beads.

Example 30 Detection of Kinase-Mediated Phosphorylation of PeptideSubstrates Bound to Streptavidin-Polystyrene Particles Using a BindingSolution of the Present Invention

Streptavidin-polystyrene beads (4.0-4.9 μM) were charged with abiotinylated synthetic peptide called crosstide. Crosstide is a peptidesubstrate for the serine/threonine kinase Akt/Protein Kinase B and is a1808 g/mol peptide with the following amino acid sequence,Gly-Arg-Pro-Arg-Thr-Ser-Ser-Phe-Ala-Glu-Gly. Beads were charged in 100mM Tris, 100 mM NaCl, pH 7.5 and washed several times in the samebuffer, following charging, before staining. The crosstide peptide onthe streptavidin polystyrene particle was then phosphorylated using 500ng of Akt/PKB kinase in 40 μL of 15 mM MOPS, pH 7.2, 18.75 mM β-glycerolphosphate, 3.75 mM EGTA, 0.75 mM sodium orthovanadate, 0.75 mM DTTsupplemented with 200 μM ATP. A control reaction was performed in whichall reaction components were added, including ATP, except the kinaseenzyme. Phosphorylation was carried out for 60 minutes at 30° C. withcontinuous rotation and stopped by incubating beads and kinase at 100°C. for 5 minutes. Beads were then washed again by incubating in 100 mMTris, 100 mM NaCl, pH 7.5 followed by staining with 1 μM Compound 2 inbuffer containing 0.05 M sodium acetate, pH 4.0, 1 μM GaCl₃, 0.5 M NaCl,and 20% 1,2 propanediol for 45 minutes. Following staining, beads werewashed in 0.05 M sodium acetate, pH 4.0, 4% acetonitrile and imagedusing a Nikon Eclipse 800 Epi-Fluorescent Microscope using OmegaOptical, Inc. filter set XF102-2 (Exciter: 560AF55; Dichroic: 595DRLP;Emitter: 645AF75). The fluorescent signal of the peptide charged beadsexposed to Akt/PKB kinase was found to be 2.2-fold higher, with nooverlap in standard deviation, than the control peptide charged beadsnot exposed to enzyme.

Example 31 Synthesis of Compound 2

A solution of 3-dimethylaminophenol (0.47 g, 3.5 mmol) and5-fluoro-5′-formyl BAPTA tetramethyl ester (1.00 g, 1.7 mmol) in 20 mLpropionic acid was heated at 110° C. for 2 hours, cooled and poured into120 mL aqueous NaOAc. The resulting purple gum was rinsed with water,dissolved in ethyl acetate, and evaporated to give 1.20 gdihydro-Rhod-5F tetramethyl ester as a red foam.

To dihydro-Rhod-5F tetramethyl ester (1.2 g, 1.5 mmol) in 1:1chloroform/methanol (40 mL) was added chloranil (0.51 g, 2.0 mmol). Thesolution was stirred overnight at room temperature then evaporated. Theresidue was purified by flash chromatography usingchloroform/methanol/acetic acid (50:5:1) as eluant to give 0.54 g ofRhod-5F tetramethyl ester as a red foam.

To Rhod-5F tetramethyl ester (0.48 g, 0.55 mmol) in dioxane (25 mL) wasadded 1 M KOH (4.4 mL, 4.4 mmol). The solution was stirred overnight,then evaporated. The residue was dissolved in 10 mL water and 50 mL of5% HCl was added. A precipitate was filtered and dried to give 275 mg ofRhod-5F free acid as a red powder. This product was converted to thepotassium salt with aqueous KOH, followed by column chromatography withwater on Sephadex LH-20 to give tripotassium salt Compound 2 as a redpowder.

Example 32 Synthesis of Compound 5 (Rhodamine BAPTA compound)

8-Hydroxyjulolidine (0.76 g, 4.1 mmol), 5-fluoro-5′-formyl BAPTAtetramethyl ester (1.16 g, 2.0 mmol) and p-TsOH (20 mg) in 20 mLpropionic acid were heated at 60° C. overnight, then cooled and pouredinto 150 mL aqueous 3 M NaOAc. A purple powder was collected byfiltration, rinsed with water, and dried to give 2.15 g ofdihydro-X-Rhod-5F tetramethyl ester as a purple powder.

To dihydro-X-Rhod-5F tetramethyl ester (2.1 g, 2.4 mmol) in 1:1chloroform/methanol (80 mL) was added chloranil (1.45 g, 5.9 mmol). Thesolution was stirred 4 hours at room temperature then evaporated. Theresidue was purified by flash chromatography usingchloroform/methanol/acetic acid (50:5:1) to give 3.0 g of X-Rhod-5Ftetramethyl ester as a red foam.

To X-Rhod-5F tetramethyl ester (3.0 g, 2.9 mmol) in dioxane (25 mL) andmethanol (25 mL) was added 1 M KOH (30 mL, 30 mmol). The solution wasstirred overnight then evaporated. The residue was dissolved in 10 mLwater and this added to 50 mL of 5% HCl. A precipitate was filtered anddried to give 500 mg of Compound 5 free acid as a purple powder. 100 mgof the free acid was converted to the potassium salt with aqueous KOH,followed by chromatography with water on Sephadex LH-20 to give 40 mg ofCompound 5 as its potassium salt, a purple powder.

Example 33 Synthesis of Quinazolinone-Labeled BAPTA (Q-BAPTA) Compounds(Compounds 7 and 23) Preparation of 5-Fluoro-Q-BAPTA (Compound 7)

a catalytic quantity of p-toluenesulfonic acid (TsOH) was added to asolution of anthranilamide (29 mg, 0.21 mmol) and5′-fluoro-5-formyl-4-hydroxy-BAPTA tetramethylester (128 mg, 0.21 mmol)in 10 mL dichloroethane/5 mL ethanol. The solution was refluxedovernight then cooled. Chloranil (57 mg, 0.23 mmol) was added. After 2hours, the solution was evaporated and the residue was purified by flashchromatography using 5% methanol/chloroform to yield 50 mg of thetetramethylester of Compound 7 as a light-amber immobile oil; m/z 711(710 calc for C₃₄H₃₄N₄O₁₂F).

To a green solution of the tetramethylester of compound 7 (50 mg, 0.07mmol) in 1:1 dioxane:methanol (5 mL), was added 1 M aqueous KOH (0.56mL, 0.56 mmol). The yellow solution was stirred overnight thenevaporated. The residue was purified with water on Sephadex LH-20,generating 53 mg of compound 7 as its potassium salt as a yellow powder;m/z (positive mode) 655 (651 calculated for C₃₀H₂₃N₄O₁₂F).

Preparation of 5,6-Difluoro-Q-BAPTA (Compound 23)

5,6-Difluoro-4′-hydroxy-5′-formyl BAPTA tetramethylester (0.100 g, 0.163mmol) and anthranilamide (0.022 g, 0.162 mmol) were dissolved in amixture of methylene chloride (10 mL) and ethanol (5 mL). TsOH (5 mg)was added and the reaction mixture was refluxed for 3 hrs. Chloranil(0.044 g, 0.18 mmol) was added to the solution. The mixture was refluxedfor 2 more hours and evaporated. The crude product was purified bypreparative TLC using 2:1 chloroform-ethyl acetate as eluant. The maincomponent (R_(f)=0.5) was isolated with ethyl acetate, which solutionwas evaporated to give 5,6-difluoro-Q-BAPTA tetramethylester as acolorless powder (0.029 g, 24%).

5,6-Difluoro-Q-BAPTA tetramethylester (0.027 g, 0.037 mmol) wasdissolved in a mixture of 1 mL of methanol and 1 mL of dioxane. 1 M KOH(1 mL) was added to the solution and the reaction mixture was keptovernight at room temperature. Volatiles were evaporated, the crudeproduct was redissolved in water and purified on a Sephadex LH-20column, eluting with water. The product was lyophilized to give 0.021 gof 5,6-difluoro-Q-BAPTA potassium salt (Compound 23) as a yellow powder(R═CH₂CO₂K).

Example 34 Synthesis of (borapolyazaindacene BAPTA) Compounds (Compound8 and 24) Preparation of BODIPY FL Dye BAPTA-5F (Compound 8)

To a cold solution of 5-fluoro-BAPTA tetramethylester (1.00 g, 1.82mmol) in 9 mL acetic anhydride was added 70% nitric acid (0.15 mL, 2.3mmol). After 10 minutes, the reaction solution was poured into 30 mLaqueous NaOAc then saturated aqueous sodium bicarbonate was added. Themixture was extracted with chloroform (2×30 mL). The extract was washedwith brine, dried over sodium sulfate, and concentrated to an amberresidue. This was purified by flash chromatography using ethylacetate/hexanes to give 0.43 g of 5-nitro-5′-fluoro-BAPTA,tetramethylester as a yellow powder.

To 5-nitro-5′-fluoro-BAPTA, tetramethylester (0.43 g, 0.72 mmol) in 1:1methanol/dioxane (10 mL) was added 1 M KOH (5.8 mL, 5.8 mmol). Thesolution was stirred overnight then evaporated. The residue wasdissolved in 10 mL water, and the pH lowered to 2 with aqueous HCl. Aprecipitate was collected and dried to give 0.31 g of5-nitro-5′-fluoro-BAPTA free acid as a yellow powder.

A solution of 5-nitro-5′-fluoro-BAPTA free acid (0.31 g, 0.58 mmol) in30 mL methanol was shaken over 10% Pd/carbon (0.15 g) under 38 psihydrogen gas for 6 hours, then filtered and evaporated to give 0.26 g of5-amino-5′-fluoro-BAPTA free acid as a colorless powder.

BODIPY FL dye free acid (Molecular Probes, Inc. D-2183, 27 mg, 0.09mmol) in 5 mL anhydrous THF was treated with an oxalyl chloride (0.20mmol) and diisopropylethylamine (DIEA, 0.20 mmol) under argon. After 15minutes, the solution was evaporated. The residue was dissolved in 3 mLanhydrous dioxane, and this solution was slowly added to a solution of5-amino-5′-fluoro-BAPTA free acid (50 mg, 0.10 mmol) in 5 mL water thathad been pH-adjusted to pH=9.5 with sodium carbonate. This solution wasstirred overnight then evaporated to near dryness. This solution waspurified with water elution on Sephadex LH-20 to yield 41 mg of BODIPYFL Dye BAPTA-5F (Compound 8), sodium salt as an orange powder.

Preparation of BODIPY FL Dye-EDA-BAPTA (Compound 24)

To a solution of 5-amino-BAPTA free acid (853 mg, 1.74 mmol) in water(50 mL) and con. HCl (1.0 mL), thiophosgen (10 mL) in chloroform (50 mL)was added and vigorously stirred at rt for 8 h. The organic solvent wasevaporated, and the precipitate was collected by a centrifugal. Thedried precipitate was redissolved in THF (20 mL), and precipitated withhexanes (200 mL). The precipitate was collected by a centrifugal anddried to give 5-isothiocyanato-BAPTA free acid (640 mg).

The pH of a solution of BODIPY FL ethylenediamine hydrochloride salt (15mg, 0.04 mmol, Molecular Probes) in 3 mL water was raised to 7.6 bydropwise addition of aqueous sodium bicarbonate. A solution of5-isothiocyanato-BAPTA free acid (22 mg, 0.04 mmol) in 2 mL dioxane wasadded. The pH was raised to 9.5 with aqueous sodium carbonate, and theorange solution was stirred at room temperature overnight. The solutionwas evaporated to 2 mL, and the this solution was purified on SephadexLH-20 using water for elution to give 17 mg of BODIPY FL-EDA-BAPTAsodium salt (Compound 24) as a fine orange powder after lyophilization(R═CH₂CO₂Na).

Example 35 Synthesis of Biotinylated BAPTA Compounds (Compounds 9, 12and 18) Preparation of Biotin-BAPTA-5F (Compound 12)

A solution of 5-nitro-5′-fluoro-BAPTA, tetramethylester was reduced bycatalytic hydrogenation over 10% Pd/C in ethyl acetate. To the resulting5-amino-5′-fluoro-BAPTA, tetramethylester (0.10 g, 0.18 mmol) inanhydrous dichloromethane/THF (4:1, 5 mL) was added glutaric anhydride(40 mg, 0.36 mmol) and catalytic DMAP. The solution was stirredovernight then evaporated. The residue was purified by flashchromatography using 10% methanol/chloroform to give 0.13 g of theglutaramide of 5-amino-5′-fluoro-BAPTA, tetramethylbester as an oil.

To the glutaramide of 5-amino-5′-fluoro-BAPTA, tetramethylester (0.18mmol) in 5 mL anhydrous THF and 5 mL anhydrous acetonitrile was addedN-hydroxysuccinimidyluronium tetrafluoroborate (108 mg, 0.36 mmol).After two hours a solution of biotin ethylenediamine hydrobromide (66mg, 0.18 mmol, Molecular Probes) and DIEA (0.05 mL) in 2 mL anhydrousDMF was added. After stirring overnight, the volatiles were evaporated.The residue was triturated with water (15 mL), and the resultingprecipitate was collected, rinsed with water, and dried to give 0.10 gof biotin-BAPTA-5F tetramethylester as a gray powder.

To biotin-BAPTA-5F tetramethylester (0.10 g, 0.11 mmol) in 1:1methanol/dioxane (4 mL) was added 1 M KOH (1.0 mL, 1.0 mmol). Thesolution was stirred overnight then evaporated. The residue was purifiedon Sephadex LH-20 using water, which gave biotin-BAPTA-5F (Compound 12)potassium salt as a colorless powder after lyophilization.

Preparation of Rhod-biocytin (Compound 18)

To a 0.5 M solution of 4-(succinimidyloxycarbonyl)-rhod tetramethylester in anhydrous THF was added 1.1 equivalent ofN-t-BOC-ethylenediamine and 1.1 equivalent of DIEA. The resultingsolution was stirred for 30 minutes then evaporated. The residue waspurified by flash chromatography using chloroform/methanol/acetic acid.The purified carbonate was dissolved in dichloromethane and treated withtrifluoroacetic acid (20 equivalents). This solution was stirred 30minutes, then evaporated and dried to give the ethylenediaminecarboxamide of 4-carboxy-rhod tetra methyl ester.

To a 0.5 M solution of the ethylenediamine carboxamide of 4-carboxy-rhodtetramethyl ester in DMF was added N-t-BOC-biocytin succinimidyl ester(1.5 equivalent, described in Wilbur et al., Bioconjugate Chemistry, 11:584-98 (2000)) and DIEA (1.5 equivalent). The resulting solution wasstirred at room temperature until the TLC indicated consumption of thefluorescent starting material. The volatiles were removed in vacuo, andthe residue was purified by flash chromatography usingchloroform/methanol/acetic acid to give N-t-BOC-rhod-biocytintetramethyl ester.

A 0.5 M solution of N-t-BOC-rhod-biocytin tetramethyl ester in 1:1methanol/dioxane was treated with 12 equivalents of 1 M KOH. Theresulting solution was stirred overnight at room temperature thenevaporated to dryness. The residue was purified on Sephadex LH-20 usingwater to give Compound 18 as a red powder after lyophilization(R═CH₂CO₂K).

Preparation of Rhod-4-biotin-BAPTA (Compound 9)

A suspension of (2′-nitrophenoxy)-2-chloroethane (20.15 g, 0.10 mol),methyl (4-hydroxy-3-nitro)benzoate (21.67 g, 0.11 mol), and K₂CO₃ (27.60g, 0.20 mol) was stirred at 130° C. for 16 h, cooled to roomtemperature, and poured into ice water (1.2 L). The precipitate wasfiltered, washed with H₂O and dried to give 32.00 g of(4′-methoxycarbonyl-2′-nitrophenoxy)-2-(2″-nitrophenoxy)ethane as ayellow solid.4′-Methoxycarbonyl-2′-nitrophenoxy)-2-(2″-nitrophenoxy)ethane (20.0 g,55.2 mmol) was hydrogenated over 10% Pd/C (3.0 g) in DMF (300 mL) at 40psi for 5 h. The mixture was filtered from catalyst through Celite. Thefiltrate was evaporated and ether (100 mL) was added. The product wasfiltered and washed with ether (2×25 mL) to give 13.2 g of1′-amino-4′-methoxycarbonylphenoxy)-2-(2″-aminophenoxy)ethane as anoff-white solid.

A mixture of2′-amino-4′-methoxycarbonylphenoxy)-2-(2″-aminophenoxy)ethane (13.20 g,44 mmol), methanol (50 mL), dioxane (50 mL), and 1 M KOH (100 mL, 100mmol) was stirred at 65° C. for 5 h, then overnight at room temperature.The mixture was evaporated and the residue was suspended in H₂O (500mL). Aqueous 1 M HCl was added to pH 5.0. The precipitated product wasfiltered, washed with H₂O, and dried on a filter for 4 h, then washedwith ether (3×25 mL) to give 12.5 g of2′-amino-4′-carboxy-1′-phenoxy)-2-(2″-aminophenoxy)ethane as anoff-white solid.

Diphenyldiazomethane was prepared by vigorously stirring benzophenonehydrazone (6.66 g, 34 mmol) and yellow HgO (17.60 g, 80 mmol) in hexanes(200 mL) for 3 h. The mixture was filtered from inorganics, evaporatedand the residue was dissolved in acetone (50 mL). This solution wasadded to a suspension of2′-amino-4′-carboxy-1′-phenoxy)-2-(2″-aminophenoxy)ethane (5.76 g, 20mmol) in acetone. The mixture was stirred for 16 h at 35° C., then theexcess of diphenyldiazomethane was decomposed with AcOH (2 mL) over 2 h.The mixture was evaporated, and the crude product was purified by flashchromatography on SiO₂ using CHCl₃ as eluant to give 6.80 g of2′-amino-4′-diphenylmethoxycarbonylphenoxy)-2-(2″-aminophenoxy)ethane asan off-white solid.

A mixture of2′-amino-4′-diphenylmethoxycarbonylphenoxy)-2-(2″-aminophenoxy)ethane(8.28 g, 18.24 mmol), DIEA (16.3 mL, 94 mmol), methyl bromoacetate (35.3mL, 376 mmol), and NaI (1.50 g, 10 mmol) in MeCN (400 mL) was refluxedunder stirring for 70 h, cooled to room temperature and evaporated. Theresidue was dissolved in CHCl₃ (500 mL), washed with 1% AcOH (3×200 mL),H₂O (200 mL), sat. NaCl (200 mL), filtered and evaporated. The residuewas purified by flash chromatography on SiO₂ using a gradient of 30-40%EtOAc in hexanes as eluant to give 10.01 g of4-diphenylmethoxycarbonyl-BAPTA tetramethyl ester as a white solid.

To a solution of Vilsmeier reagent made from POCl₃ (5 mL, 50 mmol) inDMF (35 mL) was added a solution of 4-diphenylmethoxycarbonyl-BAPTAtetramethyl ester (3.71 g, 5 mmol) in DMF (15 mL). The mixture wasstirred at 40° C. for 24 h, then another portion of Vilsmeier reagent(25 mmol) was introduced and the mixture was stirred at 40° C. for 70 h.The mixture was cooled to room temperature and quickly poured into anice-sat. K₂CO₃ mixture (1200 mL). After 1 h, the precipitate wasfiltered, washed with H₂O and dried to give 3.78 g of4-diphenylmethoxycarbonyl-5′-formyl-BAPTA tetramethyl ester as acolorless solid.

A mixture of 4-diphenylmethoxycarbonyl-5′-formyl-BAPTA tetramethyl ester(2.90 g, 3.8 mmol), m-dimethylaminophenol (1.21 g, 8.8 mmol), and TsOH(100 mg,) in propionic acid (40 mL) was stirred at 68° C. for 20 h, thencooled to room temperature and poured into 3 M NaOAc (600 mL). After 1h, the precipitate was filtered, washed with water, and dried to give3.70 g of 4-diphenylmethoxycarbonyl-dihydrorhod tetramethyl ester as apurple-red solid.

A mixture of 4-diphenylmethoxycarbonyl-dihydrorhod tetramethylester(2.050 g, 2.0 mmol) and powdered chloranil (0.492 g, 2.0 mmol) in CHCl₃and MeOH (40 mL of each) was stirred for 2 h, filtered and evaporated.The residue was purified by flash chromatography on SiO₂ using agradient 5-6.5% MeOH in CHCl₃/1% AcOH as eluant to give a crude product,which was re-dissolved in CHCl₃, filtered from SiO₂, and evaporated togive 0.533 g of 4-diphenylmethoxycarbonyl-rhod tetramethyl ester as adark-purple solid.

To 4-diphenylmethoxycarbonyl-rhod tetramethyl ester (51 mg, 0.05 mmol)in dioxane (2 mL) and MeOH (1 mL) was added 1 M KOH to give pH 12.0. Themixture was stirred for 20 h, then the pH adjusted to 9.0 with 0.1 MHCl. The mixture was evaporated and the residue purified on SephadexLH-20 using H₂O as eluant. The product was lyophilized to give 26 mg of4-carboxy-rhod tetrapotassium salt as a red-purple solid.

To 4-diphenylmethoxycarbonyl-rhod tetramethyl ester (102 mg, 0.1 mmol)in CHCl₃ (10 mL) was added TFA (10 mL) and the resulting mixture wasstirred for 1 h then evaporated and co-evaporated with CHCl₃ (3×10 mL).Ether (10 mL) was added and the precipitate was filtered and washed withether (3×10 mL) to give 82 mg of 4-carboxy-rhod tetramethyl ester as adark purple solid.

To 4-carboxy-rhod tetramethyl ester (80 mg, 0.093 mmol) in DMF (2 mL)was added DIEA (0.35 mL, 2 mmol) and dryO-trifluoroacetyl-N-hydroxysuccinimide (TFA-SE, 225 mg, 1 mmol). Themixture was stirred for 2 h, then more TFA-SE (113 mg, 0.5 mmol) wasintroduced and the mixture stirred for another 16 h. The mixture wasdiluted with CHCl₃ (50 mL), washed with 1% AcOH (3×20 mL), H₂O (25 mL),sat. NaCl (50 mL), filtered and evaporated. Ether (25 mL) was added andthe precipitated product was filtered and washed with ether to give 86mg of 4-(succinimidyloxycarbonyl)-rhod tetramethyl ester as adark-purple solid.

To biotin cadaverine (34 mg, 0.077 mmol, Molecular Probes, Inc.) in DMF(1 mL) and DIEA (0.055 mL, 0.40 mmol) was added a solution of4-(succinimidyloxycarbonyl)-rhod tetramethyl ester (36 mg, 0.038 mmol).The mixture was stirred for 3 h, diluted with CHCl₃ (200 mL), washedwith 1% AcOH (3×150 mL), H₂O (100 mL), sat. NaCl (200 mL), filtered andevaporated. The residue was purified on two preparative TLC SiO₂ plates,using 12% MeOH and 2.5% AcOH in CHCl₃ as eluant to give 38 mg of4-(N-(5″-biotinylaminopentyl)aminocarbonyl)-rhod tetramethyl ester.

To 4-(N-(5″-biotinylaminopentyl)aminocarbonyl)-rhod tetramethyl ester(30 mg, 0.025 mmol) in MeOH (2 mL) and H₂O (1 mL) was added 1 M KOH togive pH 12.0. The mixture was stirred for 20 h then adjusted to pH 8.5with 0.1 M HCl. The mixture was evaporated and the residue purified onSephadex LH-20 using H₂O as eluant. The product was lyophilized to give30 mg of Compound 9 (4-(N-(5″-biotinylaminopentyl)aminocarbonyl)-rhodtetrapotassium salt) as an orange-red solid.

Example 36 Synthesis of4-(4′-(Aminophenyl)-2-ethylamino)carbonylmethyl-rhod tripotassium Salt(Compound 10)

A suspension of (2′-nitrophenoxy)-2-chloroethane (5.87 g, 29 mmol),methyl 4-hydroxy-3-nitrophenyl acetate (6.15 g, 29 mmol), and K₂CO₃(8.28 g, 60 mmol) was stirred at 120° C. for 16 h, cooled to roomtemperature, and poured into ice water (0.6 L). The precipitate wasfiltered, washed with H₂O and dried to give 4.49 g of(4′-methoxycarbonylmethyl-2′-nitrophenoxy)-2-(2″-nitrophenoxy)ethane asa yellow solid.

4′-(Methoxycarbonylmethyl-2′-nitrophenoxy)-2-(2″-nitrophenoxy)ethane(9.6 g, 25.5 mmol) was hydrogenated over 10% Pd/C (1.0 g) in DMF (250mL) at 40 psi for 16 h. The mixture was filtered from catalyst throughCelite. The filtrate was evaporated and the residue was purified byflash chromatography on SiO₂ using a gradient of 25-35% EtOAc in hexanesto give 5.53 g of(2′-amino-4′-methoxycarbonylmethylphenoxy)-2-(2″-aminophenoxy)ethane asan off-white solid.

A mixture of(2′-amino-4′-methoxycarbonylmethylphenoxy)-2-(2″-aminophenoxy)ethane(5.50 g, 17.4 mmol), methanol (40 mL), dioxane (40 mL), and 1 M KOH (35mL, 35 mmol) was stirred at 45° C. for 1 h, then overnight at roomtemperature. The mixture was evaporated and the residue was suspended inH₂O (100 mL). Aqueous 1 M HCl was added to pH 3.0. Precipitated productwas filtered, washed with H₂O, and dried to give 4.59 g of(2′-amino-4′-carboxymethyl-1′-phenoxy)-2-(2″-aminophenoxy)ethane as anoff-white solid.

Diphenyldiazomethane was prepared by vigorously stirring benzophenonehydrazone (2.94 g, 15 mmol) and yellow HgO (8.80 g, 40 mmol) in hexanes(70 mL) for 5 h. The mixture was filtered from inorganics, and thefiltrate was evaporated and the residue was redissolved in acetone (20mL). This solution was added to the solution of the2′-amino-4′-carboxymethyl-1′-phenoxy)-2-(2″-aminophenoxy)ethane (3.02 g,10 mmol) in acetone (120 mL). The resulting mixture was stirred for 16 hat 35° C., then the excess of diphenyldiazomethane was decomposed withAcOH (0.5 mL) over 2 h. The mixture was evaporated, and the crudeproduct was purified by flash chromatography on SiO₂ using 1% MeOH inCHCl₃ as eluant to give 4.44 g of(2′-amino-4′-diphenylmethoxycarbonylmethylphenoxy)-2-(2″-aminophenoxy)ethaneas an off-white solid.

A mixture of2′-amino-4′-diphenylmethoxycarbonylmethylphenoxy)-2-(2″-aminophenoxy)ethane(2.12 g, 4.5 mmol), DIEA (4.0 mL, 23.5 mmol), methyl bromoacetate (8.8mL, 94 mmol), and NaI (0.50 g, 4.7 mmol) in MeCN (90 mL) was refluxedfor 70 h, cooled to room temperature and evaporated. The residue wasdissolved in CHCl₃ (500 mL), washed with 1% AcOH (3×200 mL), H₂O (200mL), sat. NaCl (200 mL), filtered and evaporated. The residue waspurified by flash chromatography on a SiO₂ column using a gradient of30-40% EtOAc in hexanes as eluant to give 2.82 g of4-diphenylmethoxycarbonylmethyl-BAPTA tetramethyl ester as a colorlesssolid.

To a solution of Vilsmeier reagent made from POCl₃ (1.5 mL, 30 mmol) inDMF (10 mL) was added a solution of4-(diphenylmethoxycarbonylmethyl)-BAPTA tetramethyl ester (3.78 g, 5mmol) in DMF (5 mL). The mixture was stirred for 24 h then quicklypoured into an ice-sat. K₂CO₃ mixture (500 mL). The mixture wasextracted with CHCl₃, dried over MgSO₄ and evaporated. The mixture ofproducts was separated on SiO₂ using a gradient of 30-40% EtOAc inhexanes to give 1.65 g of aldehyde4-(diphenylmethoxycarbonylmethyl)-5′-formyl-BAPTA tetramethyl ester as acolorless solid.

A mixture of 4-(diphenylmethoxycarbonylmethyl)-5′-formyl-BAPTAtetramethyl ester (784 mg, 1.0 mmol), m-dimethylaminophenol (301 mg, 2.2mmol), and TsOH (20 mg, catalyst) in propionic acid (10 mL) was stirredat 65° C. for 20 h, then cooled to room temperature and poured into 3 MNaOAc (150 mL). After 1 h, the precipitate was filtered, washed withwater, and dried to give 450 mg of4-(diphenylmethoxycarbonylmethyl)-dihydrorhod tetramethyl ester as apurple-red solid.

A mixture of 4-(diphenylmethoxycarbonylmethyl)-dihydrorhod tetramethylester (420 mg, 0.43 mmol) and powdered chloranil (122 mg, 0.5 mmol) inCHCl₃ and MeOH (20 mL of each) was stirred for 3 h, filtered andevaporated. The residue was purified by flash chromatography on SiO₂using a gradient of 5-7% MeOH in CHCl₃/0.5% AcOH as eluant to give acrude product, which was redissolved in CHCl₃, filtered from SiO₂, andevaporated to give 275 mg of4-(diphenylmethoxycarbonylmethyl-5′-tetramethyl)-rhod tetramethyl esteras a dark-purple solid.

To a solution of 4-(diphenylmethoxycarbonylmethyl-5′-tetramethyl)-rhodtetramethyl ester (250 mg, 0.25 mmol) in CHCl₃ (20 mL) was added TFA (20mL) and the resulting mixture was stirred for 1 h, then evaporated andco-evaporated with CHCl₃ (3×30 mL). Ether (30 ml) was added to theresidue and the precipitate was filtered and washed with ether (3×10 mL)to give 200 mg of 4-carboxymethyl-rhod tetramethyl ester as adark-purple solid.

To 4-carboxymethyl-rhod tetramethyl ester (128 mg, 0.15 mmol) in DMF (5mL) and DIEA (0.40 mL, 2.2 mmol) was added dry TFA-SE (338 mg, 1.5mmol). The mixture was stirred for 16 h, then a solution of4-aminophenylethylamine (0.4 mL, 4 mmol) and DIEA (0.4 mL, 2.2. mmol)was introduced. The mixture was stirred for 2 h, diluted with CHCl₃ (500mL), washed with 1% AcOH (3×100 mL), sat. NaCl (2×200 mL), filtered andevaporated. Ether (25 mL) was added to the residue, and the precipitatedproduct was filtered and washed with ether to give 126 mg of4-(4′-(aminophenyl)-2-ethylamino)carbonylmethyl-rhod tetramethyl esteras a dark-red solid.

To 4-(4′-(aminophenyl)-2-ethylamino)carbonylmethyl-rhod tetramethylester (100 mg, 0.1 mmol) in dioxane (2 mL), MeOH (2 mL) and H₂O (1 mL)was added 1 M KOH to give pH 12.0. The mixture was stirred for 50 h thenthe pH was adjusted to 9.0 with 0.1 M HCl. The mixture was evaporatedand the residue was purified on Sephadex LH-20 using H₂O as eluant andthe product lyophilized to give 21 mg of Compound 10 as an orange-redsolid.

Example 37 Synthesis of BAPTA-Agarose Compounds (Compounds 13 and 14)Preparation of BAPTA-Agarose (compound 13)

A solution of 5-isothiocyanato-BAPTA free acid (65 mg, 0.12 mmol, U.S.Pat. No. 5,453,517) in 3 mL anhydrous DMF was added to a slurry of aminoagarose (50% aqueous slurry, 16 μmol amine/mL, 6 mL, 96 μmole amine,Pierce) that had been diluted with 15 mL DMF. The pH was raised to 10with DIEA (1.5 mL). The resulting light-brown mixture was stirred atroom temperature for 48 hours then centrifuged. The BAPTA-agarose(compound 13) pellet was rinsed with acetone (2×) and water (2×) thensuspended in water.

Preparation of BAPTA-5F-Agarose (Compound 14)

A solution of 5-amino-5′-fluoro-BAPTA free acid (0.26 g, 0.51 mmol) in12 mL aqueous HCl was diluted with 12 mL chloroform then treated withthiophosgene (3 mL). The orange mixture was stirred at room temperatureovernight then evaporated. The mixture was centrifuged, yielding a browngum that was dried then dissolved in 2 mL anhydrous THF. Addition of 20mL ethyl acetate gave a precipitate, which was isolated bycentrifugation to give 5-fluoro-5′-isothiocyanato-BAPTA free acid as alight gray-brown powder.

5-Fluoro-5′-isothiocyanato-BAPTA free acid (25 mg, 0.05 mmol) in 1 mLanhydrous DMF was added to 2 mL of a 50% aqueous slurry of amino agarosethat had been diluted with 5 mL DMF. The pH was raised to 10 with a fewdrops of DIEA. The light-brown mixture was stirred at room temperaturefor 48 hours then centrifuged. BAPTA-5F-agarose (Compound 14) pellet wasrinsed with acetone (2×) and water (2×) then suspended in water.

Example 38 Synthesis of Compound 15 (TAMRA-Biotin BAPTA Compound)

A solution of BAPTA-4-isothiocyanate free acid (18 mg, 0.033 mmol) in 5mL dioxane was added to a solution of 5-(and-6)-tetramethylrhodaminebiocytin (Molecular Probes Inc., 29 mg, 0.033 mmol) in 4 mL water. Theresulting pH (3.5) was raised to 10 with aqueous sodium carbonate. Theresulting red solution was stirred at ambient temperature overnight, theconcentrated in vacuo. The residue was purified by column chromatographyon Sephadex LH-20, using water as eluant. The product was lyophilized togive TAMRA-biotin-BAPTA as 26 mg of red powder: LCMS m/2 726 (1452calculated for C₇₃H₈₄N₁₁O₁₇S₂).

Example 39 Synthesis of Compound 16 (Rhodamine BAPTA Compound)

5-Formyl-5′-nitro-BAPTA tetramethyl ester (200 mg, 0.33 mmol) and8-hydroxyjulolidine (125 mg, 0.66 mmol) in 5 mL propionic acid washeated under nitrogen at 70° C. for 1 hour, cooled to room temperatureand poured into 30 mL concentrated potassium acetate solution. Themixture was extracted with chloroform then washed with brine, dried oversodium sulfate, and evaporated to a red oil that was purified by flashchromatography using ethyl acetate/hexanes to give 0.225 g ofdihydro-X-Rhod-5N tetramethyl ester as a yellow foam.

To dihydro-X-Rhod-5N tetramethyl ester (0.12 g, 0.12 mmol) in 1:1chloroform/methanol (5 mL) was added chloranil (40 mg, 0.16 mmol). Thesolution was stirred overnight, diluted with 50 mL chloroform, washedwith brine, dried over sodium sulfate, and evaporated. The residue waspurified by flash chromatography using 15% methanol/chloroform to give63 mg of X-Rhod-5N tetramethyl ester as a purple powder.

To X-Rhod-5N tetramethyl ester (0.11 g, 0.11 mmol) in 5 mL methanol wasadded 2 M KOH (0.6 mL, 1.2 mmol). The solution was stirred at roomtemperature overnight, then evaporated. The residue was dissolved inwater (5 mL) and the pH lowered to 2 with 2 M HCl. A precipitate wascollected by centrifugation, dissolved in fresh aqueous KOH andprecipitated with aqueous HCl. This procedure was repeated five times togive 90 mg of Compound 16 free acid as a purple powder.

Example 40 Synthesis of 4-Hydroxy-5-benzothiazolyl-BAPTA (Compound 17)

A solution of 4-hydroxy-5-formyl-5′-methyl BAPTA, tetramethylester (0.40g, 0.68 mmol) and 2-aminothiophenol (75 mg, 0.70 mmol) in DMSO (5 mL)was heated at reflux for 15 minutes. After cooling the yellow solutionwas diluted with 50 mL water. A yellow precipitate was filtered anddried, then purified by flash chromatography using ethyl acetate/hexanesto give 0.22 g of 4-hydroxy-5-benzothiazolyl-BAPTA tetramethylester as ayellow foam.

To 4-hydroxy-5-benzothiazolyl-BAPTA tetramethylester (0.21 g, 0.30 mmol)in 1:1 methanol/dioxane (10 mL) was added 1 M KOH (3.0 mL, 3.0 mmol).The solution was stirred for 3 hours then evaporated. The residue waspurified on Sephadex LH-20 using water as eluant to give 0.13 g ofcompound 17 as a yellow-green powder (R═CH₂CO₂K).

Example 41 Synthesis of 4′-Carboxymethyl-4-methoxy-rhod, potassium salt(Compound 19)

A suspension of (4′-methoxy-2′-nitrophenoxy)-2-chloroethane (11.29 g,48.7 mmol), methyl 4-hydroxy-3-nitrophenylacetate (10.80 g, 51.2 mmol),and K₂CO₃ (13.80 g, 100 mmol) was stirred at 130° C. for 4 h, cooled toroom temperature, and poured into ice water (0.8 L), allowed tocoagulate for 2 days. The precipitate was filtered, washed with H₂O anddried to give 15.1 g of(4′-methoxycarbonylmethyl-2′-nitrophenoxy)-2-(4″-methoxy-2″-nitrophenoxy)ethaneas a yellow solid.

(4′-Methoxycarbonylmethyl-2′-nitrophenoxy)-2-(4″-methoxy-2″-nitrophenoxy)ethane(15.0 g, 43.3 mmol) was hydrogenated over 10% Pd/C (2.0 g) in CH₂Cl₂(250 mL) at 45 psi for 16 h. The mixture was filtered through Celite.The filtrate was evaporated and the residue was treated with ether (200mL). The precipitate was filtered and washed with ether (3×25 mL) togive 11.21 g of(2′-amino-4′-methoxycarbonylmethylphenoxy)-2-(2″-amino-4″-methoxyphenoxy)ethaneas off-white solid.

A mixture of(2′-amino-4′-methoxycarbonylmethylphenoxy)-2-(2″-amino-4″-methoxyphenoxy)ethane(8.65 g, 25 mmol), methanol (80 mL), dioxane (80 mL), and 1 M KOH (50mL, 50 mmol) was stirred at 60° C. for 1 h, then overnight at roomtemperature. The mixture was evaporated and the residue was suspended inH₂O (300 mL). Aqueous 1 M HCl was added to pH 4.0. The precipitate wasfiltered, washed with H₂O, and dried to give 6.84 g of(2′-amino-4′-carboxymethyl-1′-phenoxy)-2-(2″-amino-4″-methoxyphenoxy)ethaneas off-white solid.

Diphenyldiazomethane was prepared by vigorously stirring benzophenonehydrazone (5.88 g, 30 mmol) and yellow HgO (17.60 g, 80 mmol) in hexanes(150 mL) for 6 h. The mixture was filtered from inorganics, filtrate wasevaporated and the residue was re-dissolved in acetone (40 mL). Thissolution was added to the solution of(2′-amino-4′-carboxymethyl-1′-phenoxy)-2-(2″-amino-4″-methoxyphenoxy)ethaneacid (6.64 g, 20 mmol) in acetone (200 mL). The resulting mixture wasstirred for 48 h at 35° C., evaporated and the residue was suspended inCHCl₃. To the suspension was added AcOH (4 mL) to decompose the excessreagent and the mixture was stirred for 2 h, then evaporated, and thecrude product was purified by flash chromatography on SiO₂ using 0.5%MeOH in CHCl₃ as eluant to give(2′-amino-4′-diphenylmethoxycarbonylmethylphenoxy)-2-(2″-amino-4″-methoxyphenoxy)ethane,7.81 g (78%) as an off-white solid. A mixture of diamine(2′-amino-4′-diphenylmethoxycarbonylmethylphenoxy)-2-(2″-amino-4″-methoxyphenoxy)ethane(4.62 g, 9.3 mmol), DIEA (52 mL, 300 mmol), methyl bromoacetate (19 mL,200 mmol), and NaI (0.75 g, 5 mmol) in MeCN (150 mL) was refluxed understirring for 70 h, cooled to room temperature and evaporated. Theresidue was dissolved in CHCl₃ (400 mL), washed with 1% AcOH (3×200 mL),H₂O (200 mL), sat. NaCl (2×200 mL), filtered and evaporated. The residuewas purified by flash chromatography on SiO₂ using a gradient of 25-40%EtOAc in hexanes as eluant to give 3.01 g of4-diphenylmethoxycarbonylmethyl-4′-methoxy-BAPTA tetramethyl ester as acolorless solid.

To a solution of Vilsmeier reagent made from POCl₃ (0.28 mL, 3 mmol) inDMF (2 mL) was added a solution of4-diphenylmethoxycarbonylmethyl-4′-methoxy-BAPTA tetramethyl ester (762mg, 1 mmol) in DMF (2 mL). The mixture was stirred for 2 h, then wasquickly poured into an ice-sat. K₂CO₃ mixture (50 mL). The mixture wasextracted with CHCl₃ (7×20 mL), dried over MgSO₄ and evaporated. Themixture of products was separated by column chromatography on SiO₂ (4×35cm bed) using a gradient of 30-45% EtOAc in hexanes to give 760 mg of4-diphenylmethoxycarbonylmethyl-5′-formyl-4′-methoxy-BAPTA tetramethylester as a colorless solid.

A mixture of 4-diphenylmethoxycarbonylmethyl-5′-formyl-4′-methoxy-BAPTAtetramethyl ester (1.58 g, 2.0 mmol), m-dimethylaminophenol (602 mg, 4.4mmol), and TsOH (50 mg, catalyst) in propionic acid (20 mL) was stirredat 65° C. for 20 h, then cooled to room temperature and poured into 3 MNaOAc (300 mL). After 1 h, the precipitated product was filtered, washedwith water, and dried to give 2.00 g of4-diphenylmethoxycarbonylmethyl-5′dihydrorhod tetramethyl ester as apurple-red solid.

A mixture of compound4-diphenylmethoxycarbonylmethyl-4′-methoxy-5′-dihydrorhod tetramethylester (2.00 g, 1.9 mmol) and powdered chloranil (0.50 g, 2 mmol) inCHCl₃ and MeOH (50 mL of each) was stirred for 4 h, filtered andevaporated. The residue was purified by flash chromatography on SiO₂using a gradient of 5-7% MeOH in CHCl₃/0.5% AcOH to give a crudeproduct, which was redissolved in CHCl₃, filtered from SiO₂, andevaporated to give 480 mg of4-(diphenylmethoxycarbonylmethyl)-4′-methoxy-rhod, tetramethyl ester asa dark-purple solid.

To a solution of 4-(diphenylmethoxycarbonylmethyl)-4′-methoxy-rhod,tetramethyl ester (45 mg, 0.04 mmol) in dioxane (1 mL), MeOH (2 mL) andH₂O (2 mL) was added 1 M KOH to pH 12.0. The mixture was stirred for 50h, then pH was adjusted to 9.0 with 0.1 M HCl. The mixture wasevaporated and the residue was purified on Sephadex LH-20 column (2.6×90cm bed) using H₂O as eluant and lyophilized to give 12 mg of Compound 19as a red solid (R═CH₂CO₂K).

Example 42 Synthesis of Compound 20 Containing a DTPA Metal-ChelatingMoiety

BODIPY® TR cadaverine, Molecular Probes D-6251, 10 mg, 0.019 mmol wasdissolved into a mixture of(S)-1-p-isothiocyanatobenzyldiethylenetriaminepentaacetic acid (DTPAisothiocyanate, Molecular Probes I-24221, 10 mg, 0.019 mmol) in 2 mLwater. The pH was raised to 10 with aqueous sodium carbonate. Theresulting blue solution was stirred at room temperature for two days,then concentrated in vacuo. The residue was purified by columnchromatography on Sephadex LH-20 using E-pure water as eluant to give 2mg of Compound 20 as a purple powder.

Example 43 Synthesis of Compound 21 Containing a DTPA Metal-ChelatingMoiety

For the synthesis of carbamate 21a a solution of penta-t-butyl1-(S)-(p-aminobenzyl)-diethylenetriaminepentaacetate (prepared accordingto the published procedure of Donald T. Corson & Claude F. Meares.Bioconjugate Chem., 11(2): 292-299 (2000), 0.800 g, 1.03 mmol) in 20 mLof methylene chloride was added 1 mL of pyridine followed by theaddition of a solution of the acid chloride of N—CBZ-6-aminohexanoicacid (0.290 g, 1.02 mmol) in 5 mL of methylene chloride. The reactionmixture was stirred overnight at room temperature and concentrated invacuo. The residue was dissolved in 100 mL of ethyl acetate and theresulting solution was washed with 10% HCl (2×30 mL), water (30 mL),brine (30 mL) and dried over sodium sulfate. The solution wasconcentrated and put on a silica gel column (packed with ethyl acetate).The column was eluted with ethyl acetate to remove impurities then thedesired product was eluted with 10:1 chloroform-methanol. Pure fractionswere combined and the solvent evaporated to give amide 21a (0.54 g, 54%)as a viscous oil.

For the synthesis of amino acid 21b, the carbamate 21a (0.700 g, 0.683mmol) was dissolved in 10 mL of TFA. The reaction mixture is kept for 3days at room temperature. Volatiles were evaporated and the residue wasre-evaporated twice from toluene, leaving a viscous oil. The oil wasstirred with ethyl acetate until it solidified. The resulting solid wasfiltered and dried to give the amino acid 21b (0.400 g, 96%).

For the synthesis of Compound 21, the amino acid 21b (0.090 g, 0.147mmol) was suspended in 10 mL of water. The pH was adjusted to ˜8 using 1M KOH. The resulting solution was added to a solution of BODIPY® TMR-X,SE, Molecular Probes D-6117, 0.03 g, 0.049 mmol in 5 mL of DMF. Thereaction mixture was stirred overnight at room temperature. The pH wasmonitored and adjusted to ˜8 during the first 2 hrs. The solution wasevaporated and the residue re-dissolved in water and purified onSephadex LH-20 using water for elution. The combined product fractionswere concentrated to ˜3 mL then lyophilized to give 0.061 g of Compound21 as a red powder.

Example 44 Synthesis of Compound 22 Containing a DTPA Metal-ChelatingMoiety

BODIPY® FL EDA, Molecular Probes D-2390, 7 mg, 0.019 mmol was dissolvedinto a mixture of DTPA isothiocyanate, Molecular Probes I-24221, 10 mg,0.019 mmol in 2 mL water. The pH was raised to 10 with aqueous sodiumcarbonate. The resulting orange solution was stirred at room temperaturefor 3.5 hours, then evaporated. The residue was purified by on SephadexLH-20 using water as eluant to give 29 mg of Compound 22 as an orangepowder.

Example 45 Synthesis of Compound 25 A BAPTA-Biotin

To a solution of biotin-cadaverine (21 mg, 0.047 mmol, Molecular Probes)in 2 mL water was added 2 drops saturated sodium carbonate solution. Asolution of 5-isothiocyanato-BAPTA free acid (25 mg, 0.047 mmol) in 3 mLdioxane was added. The reaction pH was raised to 9.5 with more sodiumcarbonate solution, and the solution was stirred overnight at ambienttemperature. The volatiles were removed in vacuo and the residue waspurified by chromatography on Sephadex LH-20 using water as eluant togive compound 25 as 40 mg of a pale brown powder (R═CH₂CO₂Na).

Example 46 Synthesis of Compound 26 A Rhod-BAPTA-BAPTA

To a solution of the ethylenediamine carboxamide of 4-carboxy-rhodtetramethyl ester (as in example 34, 0.04 mmol) in 1:1 water:dioxane (5mL) was added a solution of 5-isothiocyanato-BAPTA free acid (24 mg,0.044 mmol) in 1:1 water:dioxane (8 mL). The pH was raised to 8.5 byaddition of aqueous sodium bicarbonate. The resulting red solution wasstirred at ambient temperature overnight, then concentrated in vacuo andpurified by column chromatography on Sephadex LH-20 using water aseluant to give the intermediate tetramethyl ester tetracarboxylate as 11mg of red powder. To a solution of this intermediate (0.007 mmol) in 1.4mL water was added 1 M KOH (0.07 mmol). After 3 hours the pH (13) waslowered to 9 with aqueous acetic acid, followed by concentration invacuo. The resulting residue was purified by column chromatography onSephadex LH-20 using water as eluant to afford compound 26 as a redpowder (R═CH₂CO₂K).

Example 47 Synthesis of Compound 27a-i

The synthesis of 27a will serve to illustrate the synthetic method usedto make compounds 27a-27f. Oxalyl chloride (18 μL, 0.20 mmol) was addedto a solution of4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid(Molecular Probes B-2183, 50 mg, 0.17 mmol) in 5 mL anhydrous THF,followed by diisopropylethylamine (DIEA, 35 μL, 0.20 mmol). Theresulting solution was stirred at room temperature for 15 minutes,followed by concentration in vacuo. The resulting acid chloride wasdissolved in 3 mL dry dioxane. This solution was added dropwise to asolution of 27′ (83 mg, 0.17 mmol, X═H) in 5 mL with stirring; the pHwas maintained at 9 with sodium carbonate. The resulting cloudy orangemixture was stirred for 1 hour, whereupon silica gel TLC analysisindicated formation of 27a (R_(f) 0.40, dioxane-isopropylalcohol-water-ammonium hydroxide 15:58:13:14). The volatiles wereremoved in vacuo, and the residue purified by column chromatography onSephadex LH-20 using water as eluant. Pure product fractions were pooledand lyophilized to give 27a as 99 mg of a fluffy orange powder (68%yield): ¹H NMR (D₂O) δ 7.34 (s, 1H), 6.98-6.68 (m, 8H), 6.33 (d, J=4.0Hz, 1H), 6.19 (s, 1H), 4.18 (m, 4H), 3.66 (s, 8H), 3.18 (t, 7.6 Hz, 2H),2.72 (t, 7.6 Hz, 2H), 2.41 (s, 3H), 2.13 (s, 3H); LCMS (m/z) 765 (765calcd for C₃₆H₃₈N₅O₁₁BF₂).

R¹ R² X 27a CH₃ CH₃ H 27b Ph Ph H 27c H —CH═CH—Ph H 27d H —(CH═CH)₂—Ph H27e H 2-pyrrolyl H 27f H —CH₂CH₂CO₂Na H  8 CH₃ CH₃ F

Example 48 Synthesis of Compound 28 (Dansyl BAPTA)

A solution of dansyl chloride (22 mg, 0.081 mmol) in 3 mL dioxane wasadded to a solution of 5-amino-BAPTA (40 mg, 0.081 mmol) in 1:1dioxane/water (10 mL) at pH 9 (maintained with sodium carbonate). Theresulting solution was stirred at room temperature for 1 hour, thenconcentrated in vacuo. The residue was purified by column chromatographyon Sephadex LH-20 using water as eluant to give Compound 28 as 40 mg ofa buff powder.

Example 49 Mass Spectrometry-Based Identification of Phosphoproteinsfrom 2-D gels

2-D gels are run according to standard protocols. All steps areperformed with a volume of 500 ml/gel and 50 rpm on an orbital shaker.To remove SDS the 2-D gels are fixed in 50% MeOH, 7% HAc overnight with1 change after one hour. The gels are washed the next day for 4×15 minin dH20 before staining for 2.5-3 h with the binding solution of thepresent invention. To remove unspecific staining as well as to lower thebackground, 3 washes for 1 h each in 50 mM sodium acetate, pH 4.0, 4%acetonitrile are performed. This is followed by another wash in 50 mMNaAc, 15+% 1,2-Propanediol, pH 4.0 for another hour before imaging with532 nm laser excitation and 580 nm emission filter. The wash iscontinued over night and the imaging repeated the next day.

The phosphoprotein stain is followed by SYPRO Ruby protein gel stainover night and destaining for 2-3 h in 10% MeOH, 7% HAc before imagingagain. The gel is scanned with a 473 nm laser excitation and 580 nmemission filter and spots are cut out. For destaining the spots areplaced into a 1.5 mL centrifuge tube and destained with 100 μL 50% MeOH,5% HAc, 30 min, 100 μL 0.1% TFA, 30 min, 100 μL 50% MeOH/5% HOAc, 30min, and finally dehydrated in 100 μL 100% acetonitrile (ACN), 10 min.The pieces are completely air dried before reduction and alkylation. Ifthe proteins were already reduced and alkylated before the 2-D gelelectrophoresis, the next steps can be omitted.

For the alkylation and reduction of cysteines, add enough 20 mMdithiothreitol (DTT) in 0.1 M NH₄HCO₃ in order to completely cover thedried gel pieces (˜50 μL). It may be necessary to add more as the gelpieces re-swell. Incubate at 56° C. for 1 hr. Remove the DTT solutionand add an equal volume (50 μL) of 100 mM iodoacetamide (IAAm) in 50 mMNH₄HCO₃. Incubate at room temperature in the dark for 30 min., discardthe supernatant and wash the gel pieces 2× with 100 μL 0.1 M NH₄HCO₃ for15 min. with occasional vortexing to remove excess reagents. To extractany excess reagents from the gel pieces wash with 100 μL of 0.1 MNH₄HCO₃/50% ACN for 15 min with occasional shaking. Discard thesupernatant and wash with 100% ACN. Discard the supernatant andcompletely dry the gel pieces (air).

For in gel digestion with trypsin prepare a fresh solution of 0.05 mg/mlmodified trypsin (Promega) in 50 mM NH₄HCO₃/10% ACN. Keep on ice if notto be used immediately. Add 10 μL of the fresh trypsin solution andallow the gel pieces to soak up the trypsin solution before proceedingto the next step, i.e. 10 min. Fully re-swell the gel pieces by adding20 μL 50 mM NH₄HCO₃/10% ACN (V_(TOT)=30 μL) and incubate overnight at37° C.

To extraction the peptides, terminate digestion by adding 1 μL of 10%TFA; 10 min./RT. Vortex, spin, take out the supernatant and place in an0.5 ml eppendorf tube. Add 50 μL 0.1% TFA to the pieces and incubate 30min. Shake, spin, combine this supernatant with the first one. Add 50 μL60% ACN/0.1% TFA, 30 min., shake, spin, and combine with firstsupernatant in tube. Dry the peptides in a Speed-Vac and dissolve againin 10 μL 10% ACN/0.1% TFA.

The peptide mix can be desalted and concentrated with a C18 ZipTipcolumn from Millipore or spotted directly depending on the sampleconcentration. Mix 0.5 μL matrix (5 mg/ml α-cyano 4-hydroxycinnamix acidin 50% acetonitrile, 0.1% TFA) and 0.5 μL of sample on the target. Drythe spot and analyse in the mass spectrometer.

Example 50 Covalent Labeling and Detection of Phosphoproteins withCompound 34 (Can also use Compound 39, Compound 36, Compound 42, andCompound 44) in Polyacrylamide Gels

Purified proteins (PeppermintStick™ phosphoprotein molecular weightstandards, Molecular Probes product number P33350; alpha casein; orpepsin) were separated on 13% T, 0.8% C gels by SDS-polyacrylamide gelelectrophoresis. % T is the total monomer concentration expressed ingrams per 100 ml. and % C is the percentage crosslinker. The 0.75 mm.thick, 6×10 cm. gels were subjected to electrophoresis using the Bio-Radmini-Protean 3 system according to standard procedures.

Following separation of the proteins on the gel, the gel was fixed for30 minutes in 50 ml of 50% methanol/10% acetic acid, then overnight in100 ml of fresh fixative to ensure complete removal of the SDS. Next,the gel was rinsed two times, 10 minutes each, in 100 ml deionizedwater. The gel was then incubated for 90 minutes in the dark in 50 ml of50 mM sodium acetate, pH 4, 500 mM sodium chloride, 20% acetonitrile, 1μM gallium chloride, 1 μM Compound 34 (or Compound 36, 39, 42 or 44)dye. The gel was destained in the dark two times, 10 minutes each time,in 50 ml of 50 mM NaOAc (pH 4.0). The gel was transferred to 100 mldeionized water, and then exposed to ultraviolet light (wavelength 254nm) by placing the gel directly on a UVP brand 3UV™ transilluminator for2 minutes. A parallel gel, loaded, run, fixed and rinsed as described,was incubated for 90 minutes in a binding solution of the presentinvention containing compound 2 (See, Example 2), then destained 3×30minutes in 50 ml of 50 mM NaOAc (pH 4.0). For both gels, the redfluorescent signal produced by the dye was visualized using the 532 nmexcitation line of the SHG laser on the Fuji FLA-3000G FluorescenceImage Analyzer (Fuji Photo, Tokyo, Japan) and 580 band pass emissionfilter. Phosphoprotein bands were visible in both gels.

Both gels were then washed in 60 ml of 0.1 M sodium carbonate, pH11, for1 hour to remove noncovalently bound dye. After this base treatment, thegels were rinsed in 100 ml deionized water. The gels were imaged asbefore. The gel stained with Compound 2 had no signal left on it. Thegel incubated in Compound 34 retained fluorescence localized to thephosphoprotein bands.

Example 51 A Fluorescence Quenching Assay for Measuring Phosphorylationin Solution

Fluorescently labeled phosphotyrosine peptides were combined withCompound 2, with or without gallium. Initially, 50 μL mock kinasereactions were prepared containing 50 μM labeled peptide, 100 μM ATP and10 mM Tris buffer, pH 7.5. Subsequently, the reactions were diluted 10fold to a final volume of 500 μL containing 50 mM Na Acetate, pH 4.0, 15μM Compound 2, and 15 μM GaCl₃. 300 μL of each reaction was transferredto a 96-well plate for analysis. The samples were excited at 488 nm, andemission scans were recorded from 500-650 nm. The results are shown inthe fluorescence spectra of FIG. 12. The phosphorylated peptides areshown as follows: A, pp60-Oregon Green 488 dye (OG); B, p-Abl-OG dye; C,pp60-Alexa Fluor 488 dye (A488); D, pStat3-OG dye label. Spectra withcircles represent samples without GaCl₃ addition, and spectra withsquares represent samples with GaCl₃ addition. In all cases, when GaCl₃is added to the reaction mixture, Oregon Green dye label and Alexa Fluor488 dye label emission at 520 nm is significantly quenched. The resultsdemonstrate that quenching of the 488 nm excitable compounds can be usedto specifically quantitate phosphorylation of labeled compounds withoutinterference from free ATP in solution.

Example 52 Preparation of Compound 34

To a solution of 4-(succinimidyloxycarbonyl)-rhod tetramethyl ester (30,0.20 g, 0.18 mmol) in 3:1 THF/MeCN (20 mL) was addedN-t-butoxycarbonylethylenediamine hydrochloride (43 mg, 22 mmol) anddiisoproylethylamine (DIEA, 38 μL). The resulting solution was stirredovernight at room temperature (rt), then concentrated in vacuo. Theresidue was dissolved in 40 mL chloroform and washed with 10% citricacid (1×20 mL), dried over sodium sulfate, and concentrated to givecompound 31 as 0.23 g of a red solid.

A solution of compound 31 (0.18 g, 0.18 mmol) in 20 mL dichloromethanewas treated with 5 mL trifluoroacetic acid (TFA). After 2 h at roomtemperature the volatiles were removed in vacuo, and chloroform/toluene(1:1, 10 mL) stripped from the residue, leaving compound 32 as 0.20 g ofa red solid: m/2 442.5 (885 calcd for C₄₆H₅₆N₆O₁₂).

A solution of compound 32 (0.18 mmol, 0.18 g) in 1:1 methanol/dioxane (8mL) was treated with a 1 M solution of potassium hydroxide (KOH, 2.0 mL,2.0 mmol) at rt. After stirring overnight the reaction pH was lowered to8 with aqueous citric acid, and the volatiles removed in vacuo. Theresulting red residue was purified by column chromatography on SephadexLH-20 using water as eluant. Pure product fractions were pooled andlyophilized to give compound 33 as 0.15 g of a red powder: m/z 828 (828calcd for C₄₂H₄₇N₆O₁₂).

The pH of a solution of compound 33 (30 mg, 0.03 mmol) in 5 mL water wasraised to 9.6 with aqueous sodium carbonate. A solution of4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester (MolecularProbes product 2522, 14 mg, 0.04 mmol) in 2 mL dioxane was added. After2 h the volatiles were removed in vacuo, and the residue purified bycolumn chromatography on Sephadex LH-20 using water as eluant. Pureproduct fractions were pooled and lyophilized to give compound 34 as 34mg of a red powder: m/z 1041 (1041 calcd for C₄₉H₄₂N₉O₁₃F₄).

Example 53 Preparation of Compound 36

To a solution of compound 32 (0.22 g, 0.20 mmol) in 5 mL DMF was addedDIEA (34 μL, 0.20 mmol) and a solution of 4-benzoylbenzoic acid,succinimidyl ester (Molecular Probes product 1577, 64 mg, 0.20 mmol) in5 mL DMF. After stirring overnight the volatiles were removed in vacuo,giving compound 35 as a red residue that was used immediately in thenext step.

To a red solution of compound 35 (˜0.20 mmol) in 1:1 methanol/dioxane (6mL) was added a 1 M solution of KOH (1.0 mL, 1.0 mmol). The resultinglight brown solution was stirred at rt for 3 h, and the pH was loweredto 9.5 with aqueous citric acid. The volatiles were removed in vacuo,and the resulting red residue was purified by column chromatography onSephadex LH-20 using water as eluant. Pure product fractions were pooledand lyophilized to give compound 36 as 125 mg of a hygroscopic redpowder: m/z 1035 (1035 calcd for C₅₆H₅₁N₆O₁₄).

Example 54 Preparation of Compound 39

To a solution of compound 37 as its bis-trifluoroacetate salt (preparedby condensation of 2 eq of ethylenediamine with4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid(Molecular Probes product 6103)) (0.14 g, 0.22 mmol) in 10 mL water wasadded DIEA (35 μL, 0.20 mmol). A solution of4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester (MolecularProbes product 2522, 73 mg, 0.22 mmol) in 8 mL dioxane was addeddropwise with stirring over 10 minutes. After 1 h, the pH was lowered to2.5 with aqueous HCl, followed by concentration in vacuo. The residuewas purified by column chromatography on Sephadex LH-20 using water aseluant. Pure product fractions were pooled and lyophilized to givecompound 38 as 53 mg of an orange powder.

To a solution of BAPTA isothiocyanate (Molecular Probes product 14390, 4mg, 0.008 mmol) in 1 mL dioxane was added a solution of compound 38 (6mg, 0.008 mmol) in 1 mL water. The reaction pH was raised to 9.3 withaqueous sodium carbonate. After 2 h the volatiles were removed in vacuo,and the residue was purified by column chromatography on Sephadex LH-20using water as eluant. Pure product fractions were pooled andlyophilized to give compound 39 as 33 mg of a pale orange powder.Further purification was accomplished by dissolving all of compound 39in 2 mL water. The pH was lowered to 2 with aqueous HCl. The resultingprecipitate was collected by centrifugation and then suspended in 2 mLwater. Dissolution was affected by addition of one drop of saturatedaqueous sodium bicarbonate. The resulting solution was lyophilized togive compound 39 as 5 mg of an orange powder: m/z 1171 (1171 calcd forC₄₉H₄₅N₁₂O₁₃BF₆S).

Example 55 Preparation of Compound 42

To a solution of4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid(Compound 40, Molecular Probes product 6103)) (0.10 g, 0.30 mmol) in 15mL anhydrous acetonitrile under argon was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 57 mg,0.30 mmol). The resulting solution was stirred for 20 minutes, and4-aminomethylbenzophenone (74 mg, 0.30 mmol) and DIEA (52 μL, 0.30 mmol)were added. The resulting orange mixture was stirred at rt for 5 h, thenpartitioned between ethyl acetate (50 mL) and 10% citric acid (40 mL).The organic layer was washed with brine (1×20 mL), dried over sodiumsulfate, and concentrated to an amber residue. This residue was purifiedby flash chromatography on silica gel using methanol in chloroform aseluant to give compound 41 as 54 mg of an amber powder: m/z 529 (520calcd for C₂₉H₂₆N₃O₄BF₂).

To a solution of compound 41 (53 mg, 0.10 mmol) in 7 mL anhydrous THFunder argon was added oxalyl chloride (18 μL, 0.20 mmol) and 3 drops ofDMF. The resulting mixture was stirred at rt for 20 minutes, thenconcentrated in vacuo. The residue was dissolved in 6 mL anhydrousdioxane, and the resulting solution was added dropwise to a solution of5-amino-BAPTA free acid (49 mg, 0.10 mmol) in 6 mL aqueous sodiumcarbonate at pH 9-9.5. After stirring for 1.5 h, the reaction pH waslowered to 7.2 with aqueous citric acid, and concentrated in vacuo. Theresulting residue was purified by column chromatography on SephadexLH-20 using water as eluant. Pure product fractions were pooled andlyophilized to give compound 42 as 15 mg of an orange powder: m/z 999(999 calcd for C₅₁H₄₅N₆O₁₃BF₂).

Example 56 Preparation of Compound 44

The pH of a solution of the Compound 43 (prepared by condensingcadaverine with 4′-carboxy-fluo-4, tetramethyl ester followed bysaponification) (12 mg, 0.012 mmol) in 3 mL water was raised to 10.3with aqueous sodium carbonate. A solution of 4-benzoylbenzoic acid,succinimidyl ester (Molecular Probes product 1577, 10 mg, 0.030 mmol) in2 mL dioxane was added. After stirring at rt overnight, the volatileswere removed in vacuo. The residue was purified by column chromatographyon Sephadex LH-20 using water as eluant. Pure product fractions werepooled and lyophilized to give compound 44 as 14 mg of an orange powder:m/z 1074 (1074 calcd for C₅₆H₄₆N₄O₁₆F₂).

The reagents employed in the preceding examples are commerciallyavailable or can be prepared using commercially availableinstrumentation, methods, or reagents known in the art or whosepreparation is described in the examples. It is evident from the abovedescription and results that the subject invention is greatly superiorto presently available methods for labeling phosphorylated targetmolecules in a biological sample, as an unprecedented 500-1000 foldconcentration range of phosphorylated target molecules can be detected.The subject invention overcomes the shortcomings of the currently usedmethods by allowing labeling as well as isolation of phosphorylatedtarget molecules in a simple procedure that has increased sensitivity.It is appreciated that the methods of the present invention providelabeling of phosphorylated target molecules in solution or immobilizedand that the phosphate-binding compounds can be either immobilized or insolution, allowing for identification of enzymes responsible forphosphorylation of these target molecules. The examples are not intendedto provide an exhaustive description of the many different embodimentsof the invention. Thus, although the foregoing invention has beendescribed in extensive detail by way of illustration and example forpurposes of clarity for understanding, those of ordinary skill in theart will readily realize that many changes and modifications can be madethereto without departing from the spirit or scope of the appendedclaims.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. A binding solution comprising: a) a phosphate-binding compound havingformula (A)m(L)n(B) wherein A is a chemical moiety, L is a linker, B isa metal-chelating moiety, m is an integer from 1 to 4 and n is aninteger from 0 to 4; and b) a salt comprising metal ions.
 2. The bindingsolution of claim 1, further comprising an acid.
 3. The binding solutionof claim 1, wherein the metal ions are a cationic transition metal. 4.The binding solution of claim 1, wherein said linker comprises:—C(O)NH—.
 5. The binding solution of claim 1, wherein the chemicalmoiety is a label.
 6. The binding solution of claim 5, wherein the labelis a dye selected from the group consisting of a benzofuran, aquinazolinone, a xanthene, an indole, a benzazole and aborapolyazaindacene.
 7. The binding solution of claim 5, wherein thelabel is selected from the group consisting of a dye, an enzyme and ahapten.
 8. The binding solution of claim 1, wherein m is 1 and n is 1.9. The binding solution of claim 2, wherein the acid is acetic acid. 10.The binding solution of claim 2, wherein the acid is present at aconcentration of 1%-20%.
 11. The binding solution of claim 1, whereinthe binding solution has a pH about 3 to about pH
 6. 12. The bindingsolution of claim 1, further comprising a buffering agent.
 13. Thebinding solution of claim 12, wherein the buffering agent is selectedfrom the group consisting of salts of formate, acetate,2-(N-morpholino)ethanesulfonic acid, imidazole,N-(2-hydroxyethyl)piperazinylethanesulfonic acid, tris-(hydroxymethyl)aminomethane acetate, or tris(hydroxymethyl)aminomethane, andhydrochloride.
 14. The binding solution of claim 1, further comprising awater-miscible organic solvent.
 15. The binding solution of claim 14,wherein the water-miscible organic solvent is an alcohol.
 16. Thebinding solution of claim 15, wherein the alcohol is methanol.
 17. Thebinding solution of claim 15, wherein the alcohol is present in aconcentration of less than about 20%.
 18. The binding solution of claim1, wherein the phosphate-binding compound has the formula:(A)-[C(X)NH(CH₂)_(e)]—(B); wherein X is O and e is 0-6.
 19. The bindingsolution of claim 1, wherein the metal chelating moiety is immobilizedon a solid or semi-solid matrix.
 20. The binding solution of claim 1,wherein the metal chelating moiety is capable of binding a trivalentmetal ion.
 21. A method for detecting a phosphorylated target moleculeimmobilized on a gel comprising the following steps: immobilizing asample on a gel; contacting the gel of with a binding solutioncomprising: a) a phosphate-binding compound having formula (A)m(L)n(B)wherein A is a chemical moiety, L is a linker, B is a metal-chelatingmoiety, m is an integer from 1 to 4 and n is an integer from 0 to 4; b)a salt comprising metal ions; and, c) an acid; incubating the gel andthe binding solution for sufficient time to allow the phosphate-bindingcompound to associate with the phosphorylated target molecule; and,visualizing the phosphate-binding compound whereby the phosphorylatedtarget molecule is detected.
 22. The method of claim 21, furthercomprising contacting the gel with a fixing solution directly after theimmobilizing step.
 23. The method of claim 21, further comprising addinga second (or third) stain to the gel to detect either total protein orproteins of another class, such as glycoproteins, or both.
 24. A kit forbinding a phosphorylated target molecule in a sample, said kitcomprising: i) a binding solution comprising: a) a phosphate-bindingcompound having formula (A)m(L)n(B) wherein A is a chemical moiety, L isa linker, B is a metal-chelating moiety, m is an integer from 1 to 4 andn is an integer from 0 to 4; and b) a salt comprising metal ions; ii) atleast one of instructions, molecular weight markers that comprisephosphorylated and non-phosphorylated polypeptides, a fixing solution, adetection reagent, standards, a wash solution, a matrix, a gel, or aphosphatase or kinase substrate.
 25. The kit of claim 24, wherein thebinding solution further comprises an acid.